GS Junior Instrument Owner’s Manual
June 2012
Instrument Kit GS Junior Junior GS FLX+ XL+ GS FLX+ XLR70 GS FLX XLR70
For life science research only. Not for use in diagnostic procedures.
GS Junior Instrument Owner’s Manual
GS Junior Instrument Owner’s Manual
1 Preface ...... 5 1.1 About this Manual ...... 5 1.2 GS Junior System Documentation Set ...... 5 1.3 Revision History ...... 8 1.4 Icons ...... 9 1.5 Assistance ...... 9 1.6 Disclaimers ...... 11 1.6.1 Warranty ...... 11 1.6.2 Intended Use of the GS Junior Instrument ...... 11 1.6.3 Notice to Purchaser ...... 11 1.6.4 Open Source Software ...... 11 1.6.5 Trademarks ...... 12 1.6.6 License Disclaimers ...... 12 1.6.7 Declaration of Conformity ...... 12 1.6.8 Safety ...... 13 1.6.8.1 Normal Operation ...... 13 1.6.8.2 Specific Warnings ...... 13 1.6.8.3 Specific Cautions ...... 14 1.7 Specifications of the GS Junior System ...... 15 1.8 GS Junior System Site Requirements ...... 16 1.9 Disposal of the Instrument ...... 17 2 The GS Junior System ...... 18 2.1 Hardware Components ...... 18 2.1.1 Main Components of the GS Junior Instrument ...... 18 2.1.2 Attendant PC...... 21 2.1.3 Separate Computer Resource for Data Analysis (DataRig) ...... 21 2.1.4 Accessories ...... 21 2.2 Software Components ...... 22 2.3 Components Not Supplied...... 24 2.4 GS Junior System Installation ...... 24 3 Turning the GS Junior System On and Off, Idle Time, and Storage ...... 25 3.1 Turning On the GS Junior System ...... 25 3.2 Turning Off the GS Junior System ...... 26 3.3 Leaving the GS Junior Instrument Idle or Preparing It for Storage ...... 27 3.3.1 Idle Time ...... 27 3.3.2 Preparing the GS Junior Instrument for Storage ...... 28 3.3.3 Reactivating the GS Junior Instrument After Storage ...... 31 4 Overview of a Sequencing Run in the GS Junior System ...... 32 4.1 Continuous Instrument Operation ...... 32 4.2 Before You Begin a Sequencing Run ...... 32 4.2.1 State of the Instrument ...... 32
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4.2.2 What You Should Already Have ...... 33 4.3 Initiating and Monitoring a Sequencing Run ...... 34 5 Overview of Data Processing in the GS Junior System ...... 37
6 GS Junior Instrument Maintenance ...... 39 6.1 Sipper Tubes, Filters, and Buffer Tray Replacement ...... 39 6.1.1 Removing Used Sipper Tubes and Filters ...... 40 6.1.2 Installing New Sipper Tubes and Filters ...... 42 6.2 Maintenance Wash ...... 46 6.3 Test Sequencing Run with Control DNA Beads ...... 50 6.4 Pump Tubing and Air Filter Replacement...... 50 6.4.1 Replacing the Pump Tubing ...... 51 6.4.2 Replacing the Air Filter ...... 57 6.5 Instrument Test ...... 57 6.6 Roche Preventive Maintenance ...... 63 6.7 Reconfiguring the emPCR Shaker Adapters MV for Older TissueLyser Models ...... 63
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1 PREFACE
1.1 About this Manual
The GS Junior Instrument Owner’s Manual briefly describes the hardware and software components of the GS Junior System, tells how to maintain the instrument, and provides guidelines for troubleshooting instrument problems.
1.2 GS Junior System Documentation Set
The documentation set describing the GS Junior System comprises the documents listed in Table 1. A brief description of each document is given below. These are available electronically in printable format (PDF) as well as in e-manual format. The e-manual format offers the added convenience of point-and-click navigation, plus some special online features. All documents can be accessed and downloaded from the customer-restricted area of our web site: www.454.com/my454.
In this documentation, the phrase “GS Junior System” refers to the whole system for DNA sequencing developed by 454 Life Sciences Corp., including the GS Junior Instrument and its Attendant PC components, all the kits for the preparation, amplification and sequencing of a DNA sample, the methods to use the kits as described in the Manuals and Guides, and the software provided to process and analyze the data from sequencing Runs. Likewise, “GS FLX System” refers to our similar high-throughput system based on the Genome Sequencer FLX Instrument; and “GS FLX+ System” refers to our high-throughput system based on the GS FLX+ Instrument. The phrase “454 Sequencing System” refers to the common technology that underlies these systems. 454 Life Sciences Corporation is a Roche company.
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Manual/Guide Title
System-Wide Documents: GS Junior Instrument Owner’s Manual (this document) GS Junior System Laboratory Setup Guide GS Junior System SysAdmin Guide GS Junior System Tables of Material 454 Sequencing System – An Overview GS Junior System Research Applications Guide 454 Sequencing System Guidelines for Amplicon Experimental Design
Laboratory Method Manuals, GS Junior Titanium Series: Rapid Library Preparation Method Manual Paired End Rapid Library Preparation Method Manual – 20 kb and 8 kb Span Paired End Rapid Library Preparation Method Manual – 3 kb Span Paired End Library Preparation Method Manual – 20 kb and 8 kb Span Paired End Library Preparation Method Manual – 3 kb Span cDNA Rapid Library Preparation Method Manual Amplicon Library Preparation Method Manual emPCR Amplification Method Manual – Lib-L, Single-Prep emPCR Amplification Method Manual – Lib-L, Multiple-Prep (MV) emPCR Amplification Method Manual – Lib-A, Single-Prep emPCR Amplification Method Manual – Lib-A, Multiple-Prep (MV) Sequencing Method Manual
454 Sequencing System Software Manual, v 2.7: General Overview and Data File Formats Part A – GS Junior Sequencer Part B – GS Run Processor, GS Reporter, GS Run Browser, GS Support Tool Part C – GS De Novo Assembler, GS Reference Mapper, SFF Tools Part D – GS Amplicon Variant Analyzer
Table 1: Components of the GS Junior System documentation set
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GS Junior Instrument Owner’s Manual – describes the GS Junior Instrument and provides information on its operation, maintenance, and troubleshooting (this document). GS Junior System Laboratory Setup Guide – describes how to set up an optimal environment for the successful operation of the GS Junior System, including recommendations for the laboratory space. Installation of the GS Junior Instrument is performed by a Roche Representative. GS Junior System SysAdmin Guide – describes how to set up the computing and networking environment for the successful operation of the GS Junior System. GS Junior System Tables of Material – lists materials required for each method that are not provided with the GS Junior System or its kits. 454 Sequencing System – An Overview – provides an upper-level description of the 454 Sequencing System, the various kits and methods it includes, and the theory of operation of each method. GS Junior System Research Applications Guide – serves as a navigational tool to help the user choose the best methods to carry out experiments using the GS Junior System. 454 Sequencing System Guidelines for Amplicon Experimental Design – provides guidelines and tips for the design of Amplicon sequencing experiments. Five types of Amplicon experimental designs are described, each form library preparation through data analysis. GS Junior Titanium Series Method Manuals, including: Rapid Library Preparation Method Manual – describes how to use the GS FLX Titanium Rapid Library Preparation Kit to prepare a DNA library suitable for sequencing with the GS Junior System, e.g. for shotgun sequencing. Paired End Rapid Library Preparation Method Manual - 20 kb and 8 kb Span – describes how to use the GS FLX Titanium Paired End Adaptor Set and the GS FLX Titanium Rapid Library Preparation Kit to prepare a library of Paired End DNA fragments spanning long (20 kb) or medium (8 kb) distances, and suitable for sequencing with the GS Junior System. Paired End Rapid Library Preparation Method Manual - 3 kb Span – describes how to use the GS FLX Titanium Paired End Adaptor Set and the GS FLX Titanium Rapid Library Preparation Kit to prepare a library of Paired End DNA fragments spanning short (3 kb) distances, and suitable for sequencing with the GS Junior System. Paired End Library Preparation Method Manual - 20 kb and 8 kb Span – describes how to use the GS FLX Titanium Paired End Adaptor Set and other reagents to prepare a library of Paired End DNA fragments spanning long (20 kb) or medium (8 kb) distances, and suitable for sequencing with the GS Junior System (legacy method). Paired End Library Preparation Method Manual - 3 kb Span – describes how to use the GS FLX Titanium Paired End Adaptor Set and other reagents to prepare a library of Paired End DNA fragments spanning short (3 kb) distances, and suitable for sequencing with the GS Junior System (legacy method). cDNA Rapid Library Preparation Method Manual – describes how to use a cDNA preparation kit in conjunction with the GS FLX Titanium Rapid Library Preparation Kit to prepare a DNA library suitable for sequencing with the GS Junior System, for cDNA / transcriptome sequencing. Amplicon Library Preparation Method Manual – describes how to prepare a library of amplicons suitable for sequencing with the GS Junior System, e.g. for sequence variant detection, identification and quantitation. (See also the 454 Sequencing System Guidelines for Amplicon Experimental Design.) emPCR Amplification Method Manual - Lib-L, Single-Prep – describes how to use the GS Junior Titanium emPCR Kit (Lib-L) to carry out a preparation of clonally amplified DNA fragments from an appropriately prepared shotgun, Paired End, or cDNA library, in a bead-immobilized form suitable for sequencing with the GS Junior System.
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emPCR Amplification Method Manual - Lib-L, Multiple-Prep (MV) – describes how to use the GS FLX Titanium MV emPCR Kit (Lib-L) and the GS FLX Titanium emPCR Breaking Kit, LV/MV, 12 pcs, to carry out up to eight parallel preparations of clonally amplified DNA fragments from appropriately prepared shotgun, Paired End, or cDNA libraries, in a bead-immobilized form suitable for sequencing with the GS Junior System. emPCR Amplification Method Manual - Lib-A, Single-Prep – describes how to use the GS Junior Titanium emPCR Kit (Lib-A) to carry out a preparation of clonally amplified DNA fragments from an appropriately prepared Amplicon DNA library, in a bead-immobilized form suitable for sequencing with the GS Junior System. emPCR Amplification Method Manual - Lib-L, Multiple-Prep (MV) – describes how to use the GS FLX Titanium MV emPCR Kit (Lib-A) and the GS FLX Titanium emPCR Breaking Kit, LV/MV, 12 pcs, to carry out up to eight parallel preparations of clonally amplified DNA fragments from appropriately prepared Amplicon DNA libraries, in a bead-immobilized form suitable for sequencing with the GS Junior System. Sequencing Method Manual – describes how to use the GS Junior Instrument in conjunction with the GS Junior Titanium Sequencing Kit and the GS Junior Titanium PicoTiterPlate Kit to determine the sequence of a properly prepared and amplified DNA library. 454 Sequencing System Software Manual – describes the data acquisition, data processing, and data analysis software used to transform raw data from a sequencing Run, or a set of sequencing Runs, into the final output of the GS Junior System (or the GS FLX or GS FLX+ Systems).
1.3 Revision History
Revision Date Instrument Version Chemistry Software Version May 2010 GS Junior GS Junior Titanium 2.5.00 September 2010 GS Junior GS Junior Titanium 2.5.00-p1 November 2011 GS Junior GS Junior Titanium 2.5.00-p1 March 2012 GS Junior GS Junior Titanium 2.7.00 June 2012 GS Junior GS Junior Titanium 2.7.00
Every effort has been made to ensure that all the information contained in the GS Junior System documentation was correct at the time of release. However, 454 Life Sciences Corporation and Roche Diagnostics GmbH reserve the right to make corrections, clarifications, updates, or any other changes deemed necessary, for any reason, without advance notice.
No part of the GS Junior System documentation may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without express written permission. Questions or comments regarding the contents of this manual can be directed to your Roche Representative or to the customer support address below:
454 Life Sciences Corporation 1 Commercial St. Branford, CT USA 06405
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1.4 Icons
Various precautionary statements are presented in the GS Junior System documentation. Such statements and other items of special interest are highlighted with the following icons:
Warning: Indicates the possibility of severe or fatal injury to the user or other persons, or damage to a system component, if the precautions or instructions are not observed.
Danger: Indique un risque de blessure sévère ou fatale à l’utilisateur ou à d’autres personnes, ou un risque d’endommager une composante du système, si les précautions ou instructions fournies ne sont pas suivies.
Caution: Highlights information that is critical for optimal performance of the system. May also indicate that loss of data or invalid data could occur if the precautions or instructions are not observed.
Attention: Souligne de l’information critique pour la performance optimale du système. Peut aussi indiquer qu’une perte de données ou des données invalides pourraient résulter si les précautions ou instructions fournies ne sont pas suivies.
Note: Identifies items of general interest and additional information about the topic or procedure being described.
Note: Identifie de l’information générale ou additionnelle sur un sujet donné.
All GS Junior System Manuals, Guides and Bulletins are available for download in PDF format and in e-manual format from the customer-restricted access area of our web site: www.454.com/my454.
1.5 Assistance
Prior to and during instrument setup, your Roche Representative will provide system support services, including a discussion of clean laboratory installation and procedures, sample handling/tracking, and data management. These services will help ensure your success with the GS Junior System. Please contact your Roche Representative for more details.
If you have questions or experience problems with the GS Junior System, please call, write, fax, or e-mail us.
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When calling for assistance, be prepared to provide the serial number of your GS Junior Instrument and/or lot number of the kit(s) you are using. The instrument’s serial number is located on the label found on the back of the instrument.
If you are located in… Please contact Roche Applied Science Technical Support via:
Phone: USA or Canada e-mail: [email protected] Region ...... (toll free) 1-800-262-4911
Phone: Europe, Middle East, or Region ...... +49-8856-60-6457 e-mail: [email protected] Africa ...... or (toll free) +800SEQUENCE
Phone: Argentina ...... +810 810 5650 Brazil ...... 0800 772 0295 Central America & Caribbean ...... 800 7624 ...... or 507 378 1245 Costa Rica ...... 8000 762 431 El Salvador ...... 503 21 13 39 35 Guatemala ...... 1 801 13 76 243 Honduras ...... 800 22 25 06 26 ...... or 504 22 25 0626 Latin America e-mail: [email protected] Nicaragua ...... 1 800 22 02 125 Panama ...... 800 7624 ...... or 507 378 1245 Chile ...... +800 373 700 Columbia ...... 01800 011 7911 Ecuador ...... +593 2 399 7291 Mexico ...... +01800 718 8853/54 Peru ...... +511 618 8990 Uruguay ...... +598 2 619 2273 Venezuela ...... +58 212 273 46 10
Phone: Region ...... (toll free) 800 820 0577 China Mainland...... 008 018 63123 China Taiwan ...... 800 966 851 China Hong Kong ...... 800 852 3686 Singapore ...... 1800 814 958 Asia Pacific e-mail: [email protected] Malaysia ...... 180 064 5619 Australia ...... 007 988 620647 Korea ...... 001 800 861 0660 Thailand ...... 1208 6101 Vietnam ...... 1800 186 10007 Philippines ...... +632 718 7506
Phone: Japan e-mail: [email protected] Region ...... +03-5443-5287
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1.6 Disclaimers
1.6.1 Warranty
Information on warranty conditions are specified in the sales contract. Contact your Roche Representative for further information. Any unauthorized modification of the instrument entails invalidation of the guarantee and service contract.
1.6.2 Intended Use of the GS Junior Instrument
The GS Junior Instrument is designed to sequence clonally amplified deoxyribonucleic acid (DNA) fragments, which are derived from double-stranded sample DNA or RNA of various origins. The instrument can sequence de novo or re-sequence an average of over 35 million bases per sequencing Run.
The GS Junior Instrument is intended for life science research applications and must be used exclusively by laboratory professionals who are trained in the correct and safe operation of this instrument and its accessories, are familiar with general laboratory techniques, and have studied the instructions for use of this instrument. If the GS Junior Instrument is used in a manner not specified in its documentation, the safety protection provided by the instrument design may be impaired.
1.6.3 Notice to Purchaser
RESTRICTION ON USE: Purchaser is only authorized to use the 454 Sequencing System Instruments with PicoTiterPlate devices supplied by 454 Life Sciences Corporation and in conformity with the operating procedures contained in the 454 Sequencing System manuals and guides.
Made in USA by 454 Life Sciences Corporation, Branford, CT, USA, a Roche company.
For life science research only. Not for use in diagnostic procedures.
1.6.4 Open Source Software
The 454 Sequencing System of Roche Diagnostics Ltd. uses open source software. Among other things, the holders of the proprietary rights grant licenses under the terms of the GNU General Public License (GPL edition 2 or above), as well as under the GNU Lesser General Public License (LGPL).
This GS Junior Instrument was designed to be operated with the unmodified software, as shipped. The user assumes full responsibility for changing any part of the open source software, which excludes any liability of Roche Diagnostics Ltd. The following disclaimer shall be valid for all parts of the software that are liable to GPL:
This program is distributed without any warranty; without even the implied warranty of merchantability or fitness for a particular purpose. See the GNU General Public License for more details (www.gnu.org/copyleft/gpl.html).
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1.6.5 Trademarks
454, 454 LIFE SCIENCES, 454 SEQUENCING, GS FLX, GS FLX TITANIUM, GS JUNIOR, EMPCR, PICOTITERPLATE, PTP, NEWBLER, REM, GTYPE, GS GTYPE, AMPLITAQ, AMPLITAQ GOLD, FASTSTART, NIMBLEGEN, SEQCAP, MAGNA PURE, and CASY are trademarks of Roche.
All other product names and trademarks are the property of their respective owners.
1.6.6 License Disclaimers
This product incorporates technology licensed from Promega Corporation that is protected under US Patents 5,583,024 (exp. 12/10/2013), 5,674,713 (exp. 10/7/2014) and 5,700,673 (exp. 12/23/2014).
Restriction on use. As a condition of sale of this product, purchaser agrees not to use the product to perform less than 1,536 sequencing reactions on a sample or samples without changing the substrate. Failure to comply with this restriction will result in an infringement of patent rights and other intellectual property rights of seller or third parties and a breach of the terms of sale of this product.
1.6.7 Declaration of Conformity
The GS Junior Instrument has been manufactured and checked in accordance with all relevant safety standards prior to leaving the factory. The instrument has been approved for use by recognized testing institutions. Conformity to recognized standards is indicated by these symbols:
The instrument meets the requirements stated in Council Directive 89/336/EEC relating to “Electromagnetic Compatibility” and Council Directive 73/23/EEC relating to “Low Voltage Equipment”.
The instrument meets the requirements of NRTL standards UL61010, CAN/CSA C22.2 No. 61010-1.
The following symbols appear on the instrument:
The CE mark on the instrument type plate expresses conformity with essential CE MARK requirements of the directives “Electromagnetic Compatibility” and “Low Voltage Equipment” relevant for this instrument.
The cTUV MARKus on the instrument type plate expresses that the product cTUV MARKus meets U.S. and Canadian safety requirements for electrical equipment for measurement, control, and laboratory use.
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Instrument Modifications: Any change or modifications made to the GS Junior Instrument, unless expressly approved in advance, in writing, by 454 Life Sciences Corporation, could void the warranty or maintenance agreement, either in part or in full.
This equipment has been tested and found to comply with the limits for a Class B digital device, pursuant to part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial or a residential environment.
1.6.8 Safety
Only trained personnel may use the GS Junior Instrument. It is essential that all users understand and observe the following safety information pertaining to installation and operation and maintenance of the GS Junior Instrument. Please ensure that all safety information is accessible to every employee working with the GS Junior Instrument.
1.6.8.1 Normal Operation
During normal operation, no one should ever need to access any internal components of the GS Junior Instrument. If, during the operation of the instrument, any of the components requires inspection, please contact your Roche Representative.
1.6.8.2 Specific Warnings
The following Warnings apply to the operation and regular (i.e. performed by the user) maintenance of the GS Junior Instrument:
Dangerous voltage: Dangerous voltages exist within the GS Junior Instrument. Users should never attempt to access the internal components of the GS Junior Instrument. Further: If maintenance is being performed, do not touch any exposed PC boards or circuits. Do not touch any electrical connections on the PC Boards or behind the camera. Users should not attempt any maintenance or service procedures. Should the GS Junior Instrument require service, please contact your Roche Representative. Failure to heed these warnings could cause personnel to come in contact with voltages that could cause severe injury or death. Instrument electrical grounding : Ensure that the wall outlet receptacle is properly wired and earth grounded. Replacing fuse: Disconnect power before replacing fuses. Débranchez le courant avant de remplacer les fusibles.
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Fuse type: For continued protection against the risk of fire, replace only with the same type and rating of fuse. Pour ne pas compromettre la protection contre les risques d'incendie, remplacer par un fusible de même type et de mêmes caractéristiques nominales. Power switch location: The main power switch is located in the right-rear of the instrument (see Section 3). L’interrupteur principal se trouve à l’arrière à droite de l’instrument (voir section 3). Heavy equipment: Use lifting aids and proper lifting technique when removing or replacing the instrument. The GS Junior Instrument weighs 55 lb (25 kg) and should be handled by two people. Delicate optical components: The optics subsystem contains components that are very precise, delicate, and expensive. Exercise great care in handling and operating any component of the optics subsystem. Constantly running fans: The camera is air-cooled by a fan (at the back of the instrument) that runs continuously. Only trained and approved service personnel should disconnect or work on any of the camera subsystem components. Never attempt to put your fingers into the air inlet/outlets in the instrument panels. Never attempt to insert any instrument, like scissors or screwdrivers, into the inlet/outlets in the instrument panels. If the fan stops or it sounds like something is caught in the fan, contact your Roche Representative immediately. Camera face: Always be extremely careful when working near the camera face. Never touch the camera face with anything other than lens paper. DO NOT USE KIMWIPES OR PAPER TOWELS TO CLEAN THE CAMERA FACE. Liquid spills: Take care of any spills near or around the camera immediately. Call your Roche Representative immediately if you suspect that fluid has lodged behind or underneath the camera. Camera door: Do not open or exert any pressure on the camera door while the camera and/or the fluidics are operating. Do not apply excess pressure to the camera face (beyond normal closing of the camera door). Do not disassemble the camera door. If service is required, contact your Roche Representative.
1.6.8.3 Specific Cautions
The following Cautions apply to the operation and regular (i.e. performed by the user) maintenance of the GS Junior Instrument:
Computer networking: Connection to computer networks contains an inherent risk of infection by viruses and worms, as well as malicious targeted attacks through the network. You MUST protect and continually update the protection of any network to which you choose to connect the GS Junior Instrument, its Attendant PC, or any data processing computer (‘DataRig’). Precautionary measures should include installing a dedicated firewall to separate the instrument network from uncontrolled networks, as well as measures to ensure that the instrument network is (and will remain) free of malicious code. Failure to heed this warning may result in irreparable damage to your data and the GS Junior Instrument.
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Fluidics subsystem: Be careful when working with the fluidics subsystem. Specifically: NEVER let the reagent inlet tubing lines of the sipper manifold (Sipper Tubes) touch any instrument surfaces. Be careful not to touch the Sipper Tubes while performing normal procedures on the GS Junior Instrument. Always wear gloves when handling the Sipper Tubes, to avoid contamination. Keep the sipper assembly in the lowered position with each Sipper Tube in its appropriate reagent container or Pre-wash Tube (unless changing reagents or Sipper Tubes). Always empty the waste bottle before a Run, to avoid overflow spills. Improper shutdown: An improper shutdown procedure may leave the GS Junior System in an unstable state which may require a service call. Make sure to follow the procedure described in Section 3.2.
1.7 Specifications of the GS Junior System
GS Junior Instrument 41 x 34 x 53 cm (16 x 14 x 21”) (H x W x D; closed) Dimensions and clearance 66 x 34 x 53 cm (26 x 14 x 21”) (H x W x D; open) requirements Approximately 30 cm (12”) clearance in front for operator access; minimum 20 cm (8”) on each side for exhaust air, and 10 cm (4”) at the rear for the power cord. Weight 25 kg (55 lbs) 100VAC – 240VAC 50/60Hz 400VA Power supply (The instrument will operate from a minimum of 85 VAC to a maximum of 264 VAC at 50/60 Hz) Fuse Type 250 VAC 4A Time-lag HT Heat output < 1440 kJ/h (< 400 W) Noise level < 65 dB(A) Backup battery Rechargeable lithium cell for the internal clock; not user-serviceable Electromagnetic emission Class B This device meets the industrial and residential immunity requirements with no Electromagnetic immunity degradation of performance (10 V/m, Criterion A). Connectivity One 1 GB Ethernet connection for the Attendant PC.
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GS Junior Attendant PC Make and Model HP Z-400 workstation Processor 1x Xeon W3550 3.066GHz with Hyper-Threading enabled Memory 12 GB ECC RAM 160 GB SATA HDD (OS/application/recovery) Storage 500 GB SATA HDD (data) Other drive DVD-RW DL drive integrated Gb NIC for external network connection (eth0) Connectivity add-on Gb NIC for instrument connection (eth1)
Uninterruptible Power Supply (UPS): The GS Junior System is not equipped with an uninterruptible power supply (UPS). It is strongly recommended that the Instrument and its Attendant PC and monitor be connected to an external UPS to avoid loss of data or the need for a service call due to improper shutdown, in the event of a transient power failure. For power failures of duration exceeding the capacity of a UPS (typically about 15 minutes), manual intervention or backup power generation would also be required to avoid the same problems. The UPS from PowerVar, model ABCE600 series, is recommended; please contact your Roche Representative for ordering information. See the GS Junior System Laboratory Setup Guide for the electrical requirements of the GS Junior System.
1.8 GS Junior System Site Requirements
Ambient temperature +15 to +30ºC (+59 to +86ºF) – air conditioning recommended
Ambient humidity 20% – 80% (non-condensing)
Ambient light < 2000 lux
For detailed information on site requirements, see the GS Junior System Laboratory Setup Guide.
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1.9 Disposal of the Instrument
Dispose of the instrument according to local and/or labor regulations.
This product complies with the Waste Electrical and Electronic Equipment (WEEE) Directive (2002/96/EC) marking requirement. The affixed product label (see below) indicates that you must not
discard this electrical/electronic product in domestic household waste.
Product Category: With reference to the equipment types in WEEE directive Annex 1, this product is classified as “Monitoring and Control instrumentation”. Do not discard it in domestic household waste.
To return unwanted products, contact your Roche Representative (see “Assistance”, above).
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2 THE GS JUNIOR SYSTEM
The GS Junior System is an automated DNA sequencing system capable of preparing, amplifying and sequencing a library of DNA fragments in a massively parallel fashion. The system provides most of the components necessary for ultra-high throughput sequencing experiments, including the GS Junior Instrument and accessories; software to generate basecalls and interpret the raw reads; and reagent kits required for library construction, clonal amplification, and sequencing.
The GS Junior Instrument Owner’s Manual provides a general description of the GS Junior Instrument, and information on how to operate, maintain and troubleshoot the instrument. For more information on the theory of operation of the GS Junior System, see the document entitled 454 Sequencing System – An Overview.
This section describes: Section 2.1: Hardware Components Section 2.2: Software Components Section 2.3: Components Not Supplied Section 2.4: GS Junior System Installation
2.1 Hardware Components
2.1.1 Main Components of the GS Junior Instrument
The main components of the GS Junior Instrument are an optics subsystem and a fluidics subsystem which occupy the left- and right-hand sides of the instrument, respectively (Figure 1). There is also a computer subsystem within the instrument which allows it to run independently once a process (e.g. a sequencing Run, after the flow calibration step) has started. However, most functions are controlled from the Attendant PC which comes with the GS Junior System (Figure 1 C; see also Section 2.1.2).
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A B
C
Figure 1: Main components of the GS Junior Instrument and System
(A) General view of the instrument with its cover closed (B) General view of the instrument with the cover and the camera door open, showing the sipper manifold in the raised position (C) Entire GS Junior System, with its Attendant PC and peripherals
The fluidics subsystem moves the sequencing reagents across the wells of a PicoTiterPlate device (PTP device), and then moves spent reagents from the PTP device to the waste bottle. The fluidics subsystem includes a Reagents cassette; a reagent sipper tube assembly and reagent tubing; a 12-position valve manifold; one main peristaltic pump; one debubbler with secondary “purging” pump; a heated PTP device holder (in the camera door); and effluent tubing leading to the waste bottle (Figure 1 B). The optics subsystem includes a cooled 6 megapixel CCD camera and a camera controller. The camera captures the light emitted from the wells of the PTP device during each step of the sequencing cycle, and sends the digital images to the computer subsystem for processing.
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The computer subsystem that resides inside the GS Junior Instrument comprises three microcontrollers that coordinate the actions of the optics and fluidics systems, temporarily store images collected in real-time on the instrument’s internal hard disk, and supervise communication with the Attendant PC. The GS Junior System comprises an Attendant PC, described in Section 2.1.2. Another visible component of the instrument is a Status Indicator LED (on the main cover, Figure 1 A), which shows at a glance the general status of the instrument.
During a sequencing Run, a PTP device containing the DNA being sequenced, constitutes the interface between the fluidics and optics subsystems (Figure 2). The side of the PTP device that is in contact with the fluidics subsystem contains microscopic (18.5 picoliter) wells in which the sequencing reactions take place. Each well is designed to contain a single, unique DNA bead carrying a clonally amplified DNA fragment. The bottom of each well is made of an optical fiber, which transmits light produced by the sequencing reaction across the thickness of the PTP device, to the camera (optics subsystem) and each well wall is lined with a metalized finish to reduce well-to-well crosstalk and signal interference.
Figure 2: Schematic showing how the GS Junior Instrument works
Sequencing reagents are pumped from the Reagents cassette to the PicoTiterPlate (PTP) area (reagent selection is done through a set of valves located inside the sipper manifold, not shown). Reagents flow across the surface of the PTP device into the reaction wells, while spent reagents flow back to the waste bottle. The light generated by the sequencing reaction (white arrows) travels through the back of the PTP device (which is constructed of optical fibers), to reach the CCD camera. Inset: Each well contains no more than one large DNA bead (sample), which is surrounded by smaller beads that carry the enzymes required for the chemiluminescence reaction.
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2.1.2 Attendant PC
The GS Junior System includes an Attendant PC which controls most of the system’s functions. The Attendant PC’s main specifications are given in section 1.7. Some of its other features include: Includes monitor, keyboard and mouse Connections to the GS Junior Instrument and (usually) to an external network via 1 GB Ethernet cables Sends all instructions to the GS Junior Instrument via a Graphical User Interface (GUI) Disk storage sufficient for over 100 sequencing Runs Linux operating system and full suite of 454 Sequencing System software Sufficient computing power to process the data of sequencing Runs and to analyze the data of multiple sequencing Runs for projects for which the GS Junior System is particularly recommended (assembly of genomes up to 12 Mbp; mapping of genomes up to 50 Mbp); for larger projects, data analysis can be carried out on a separate computer, termed a DataRig (see Section 2.1.3)
For a description of the networking requirements and recommended setup of the Attendant PC and the rest of the GS Junior System, see the GS Junior System SysAdmin Guide. For a full description of the software that comes with the GS Junior System, see the 454 Sequencing System Software Manual.
2.1.3 Separate Computer Resource for Data Analysis (DataRig)
The GS Junior System is designed such that all computing functions can be performed with the Attendant PC provided. For typical projects, the Attendant PC should be sufficient to control a sequencing Run and carry out the data analysis of another project simultaneously. However, there are circumstances where users may want or need to use separate resources. For example, if a very large project needs to be analyzed, the computing power of the Attendant PC may not allow to carry out a sequencing Run at the same time; or, a sequencing project may be too large to be analyzed on the Attendant PC altogether; or, the Attendant PC may be at a physical location that is not convenient for the person wanting to carry out an analysis.
In such cases, the data from the sequencing Run(s) can be transferred and analyzed on a separate computer loaded with the 454 Sequencing System software. Such a computer is termed a DataRig. The GS Junior System SysAdmin Guide provides guidelines for the computing power requirements for certain typical projects. For example, the Attendant PC with its single quad-core 64-bit processor and 12 GB of RAM can handle the assembly of genomes up to about 120 Mbp and mapping to genomes up to about 3 Gbp.
2.1.4 Accessories
The GS Junior Instrument comes with an Installation Kit (Table 2) which contains accessories used to handle and process the biological (DNA) sample and the sequencing reagents. The purpose and usage of these accessories are described elsewhere in this manual and in the relevant GS Junior System Method Manuals.
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Quantity Item Description 1 GS Junior Bead Counter 1 GS Junior Bead Deposition Device 1 GS Junior BDD Counterweight and Adapter Kit Includes: 1 GS Junior Bead Deposition Device Counterweight 2 Centrifuge BDD Adapters A 2 Centrifuge BDD Adapters B 1 GS Junior Sipper Maintenance Kit Includes: 10 Sipper Tubes 10 Filters 1 Buffer Tray 1 Buffer CB Sipper Tube 1 Buffer CB Sipper Filter 1 Tube De-Capping Tool 2 Data Cable CAT6 RJ-45 1 Power Cord, 6’7” unshielded 16AWG 2 Fuse, 250VAC 4A Time-lag HT 1 Zeiss wipes, box of 21 1 1000 ml sterile media bottle (used during installation)
Table 2: Contents of the GS Junior Installation Kit provided with the instrument
2.2 Software Components
The GS Junior System includes all the software required to perform sequencing experiments and fully process the resulting data. The functions of this software include: controlling the instrument for all types of Runs (pre-wash, sequencing, and maintenance) processing the raw images data into raw sequence reads assembling the raw reads into contigs and scaffolds mapping the raw reads to a reference sequence detecting, identifying and quantitating DNA Variants managing data files performing operations useful in maintenance and troubleshooting performing instrument self-diagnostic tests system administrative functions
These functions are packaged into a set of discrete software applications, as listed in Table 3. All the applications (except the GS Reporter and the GS Run Processor Manager) can be launched from a desktop icon or from a corresponding Linux command line. They are described in detail in the 454 Sequencing System Software Manual.
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Icon Application Name General Functions
Set up and management of a sequencing Run or other instrument procedure GS Junior Sequencer Acquisition of the raw images Data management
Instrument monitoring
Image processing and signal processing steps No icon GS Run Processor Produce read basecalls and per-base quality scores
No icon GS Reporter Package the output of the GS Run Processor
View and troubleshoot the results of a completed sequencing GS Run Browser Run Launch data processing (GS Run Processor Manager)
No icon GS Support Tool Package sequencing Run data to send to Roche Customer Support for further help and troubleshooting
No icon GS SFF Tools Create, manipulate and access sequencing trace data from SFF files (output of the GS Run Processor application)
GS De Novo Assembler Generate the consensus DNA sequence by assembling the reads into contigs (and scaffolds)
Generate the consensus DNA sequence by mapping the reads GS Reference Mapper to a reference sequence, and a list of high-confidence differences
GS Amplicon Variant Analyzer Allow the detection, identification and quantitation of the prevalence of sequence variants
Table 3: The software applications of the GS Junior System
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2.3 Components Not Supplied
In addition to the GS Junior Instrument, Attendant PC, and accessories, some parts of the sample preparation, sequencing, or data processing procedures require additional equipment or consumables. For more details on these, see the GS Junior System Laboratory Setup Guide, the GS Junior System SysAdmin Guide, and the GS Junior System Tables of Material. These are available in the customer access area of our web site, at www.454.com/my454.
Also, the GS Junior System supports the use of the GS FLX Titanium MV emPCR Kits (Lib-L or Lib-A) to amplify multiple libraries at the same time (see the corresponding “Multiple Prep” emPCR amplification Method Manuals). To use those kits, a pair of Shaker Adapters MV (Cat. No.: 05 618 487 001) are required. These Adaptors come pre- assembled for use with the TissueLyser II (Qiagen) but must be re-configured for previous TissueLyser models. See Section 6.7 for the re-configuration procedure.
2.4 GS Junior System Installation
At the time of this writing, GS Junior Systems are installed by a Roche Field Service Engineer. In the event that you need to move it to another location after initial installation, the basic site requirements are provided in the GS Junior System Laboratory Setup Guide (for the physical site) and the GS Junior System SysAdmin Guide (for software, and IT / networking considerations). The instrument should not be positioned so that it is difficult to operate the disconnecting device. For any questions, please contact your Roche Service Representative.
Attendant PC: The GS Junior System is provided with an Attendant PC which is tailored to the requirements of this System. Users should be aware that any alternative computer resource is not supported by Roche.
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3 TURNING THE GS JUNIOR SYSTEM ON AND OFF, IDLE TIME, AND STORAGE
3.1 Turning On the GS Junior System
The GS Junior System is designed for continuous operation and does not normally need to be turned on or off. If the system (GS Junior Instrument and Attendant PC) is off, follow the procedure below to turn it on.
1. Power on the Attendant PC (front button): a. Observe the startup sequence for errors. b. Wait for the login prompt to appear. 2. Power on the GS Junior Instrument (switch on the rear; see Figure 3): a. Wait for the LED to show solid red. b. Wait for the LED to show blinking red. c. Wait for the LED to show solid yellow. 3. Wait for the Power On Self-Test: a. Valves will click and the two pumps will turn slightly forward and backward. b. The LED will cycle green – yellow – red. c. The LED will return to solid yellow. 4. Login to the Attendant PC. 5. Double-click the “GS Junior System” icon on the desktop.
Figure 3: Location of the main switch on the GS Junior Instrument
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3.2 Turning Off the GS Junior System
Improper shutdown: An improper shutdown procedure may leave the GS Junior System in an unstable state which may require a service call.
1. Start the GS Junior Sequencer GUI if not already running. 2. If the GS Junior Sequencer application is busy, click the Abort button. 3. If you aborted, wait for the Self-Test: a. Valves will click and the two pumps will turn slightly forward and backward. b. The LED will cycle green – yellow – red. c. The LED will return to solid yellow. 4. Click the Exit button, then select the check-box that requests the instrument to stop. 5. Observe the Instrument LED: a. It will briefly change from solid yellow to blinking red, b. then change to a cycle of green – yellow – red, c. and then change to a much slower cycle of green – yellow – red – black (LED off). 6. Turn off the instrument’s main power switch (Figure 3) only when the LED is in this slow cycle pattern, with the black (LED off) between the red and green. 7. Choose System/Shutdown from the Attendant PC menu bar. a. Wait for the shutdown sequence to scroll by. b. Observe that the Attendant PC turns its power off.
If the procedure above does not function properly, you can also safely turn off the GS Junior System from the command line, using the following procedure:
1. Open a Linux window a. Type the command: ssh [email protected] b. Supply the netadmin password for the GS Junior Instrument: “netadmin” c. Type the command: halt 2. The ssh connection will close 3. Continue from step 5 of the above procedure.
NOTE: This procedure is far less desirable than the GUI method described in this section, as the GUI method provides various safeguards not available with the command line method. Use the GUI procedure whenever possible. Also, the netadmin account should not be used for any other purpose.
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Instrument emergency shutdown: During normal operation, the GS Junior Instrument should not be shut down. If a shutdown is nonetheless desired, it should be done by the orderly procedure described in this section.
In a SEVERE EMERGENCY (fire, flood, etc.), you can immediately cut off power to the system by using the MAIN POWER SWITCH on the RIGHT REAR OF THE INSTRUMENT (Figure 3). The appliance coupler of the power cord set is the main disconnect device.
3.3 Leaving the GS Junior Instrument Idle or Preparing It for Storage
3.3.1 Idle Time
Although the GS Junior System is designed for continuous operation (see Section 4.1), there are circumstances when it may be left idle. This could allow contamination growth and biofilm buildup inside your GS Junior Instrument which could result in poor or spoiled subsequent sequencing Runs and possibly require replacement of parts in the fluidics subsystem. The instructions provided in this Section should be followed when the instrument is not in continuous use. There are three situations: Up to 30 days: As soon as possible and at most within three days of a sequencing Run, the instrument should be emptied of consumables (sippers, filters, and reagents), as described in the Sequencing Method Manual. This should also be done after a maintenance wash (see Section 6.1) and after an Instrument Test (Section 6.5). The instrument can stay in this configuration for up to 30 days after the Run. Between 30 and 60 days: In addition to the removal of the sippers, filters and reagents as indicated above, if the instrument remains idle between 30 and 60 days, perform a maintenance wash immediately prior the next sequencing Run. The maintenance wash procedure is described in Section 6.1. More than 60 days: If the GS Junior Instrument is not expected to be used for a period of 60 days or more, prepare it for storage as described below.
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3.3.2 Preparing the GS Junior Instrument for Storage
1. If the instrument has already been idle for more than 3 days since the last sequencing Run, maintenance wash, or Instrument Test, perform a stand-alone maintenance wash as described in Section 6.1. 2. If present, remove and discard the Reagents cassette and the waste bottle. 3. If present, empty the bottle of Buffer CB and keep it for future use (e.g. place it in the waste position). 4. If present, remove the single long Sipper Tube and Filter and the 10 short Sipper Tubes and Filters from the sipper manifold.
When you place the GS Junior Instrument back in service, you will get all the Sipper Tubes and Filters from a GS Junior Titanium Sequencing Kit. Short Sipper Tubes and Filters can also be obtained either on their own in the GS Junior Sipper Maintenance Kit (together with a Buffer Tray); or, if you plan to run a maintenance wash, as part of the GS Junior Maintenance Wash Kit.
5. On the Attendant PC, a “Sequencing run complete” message may be displayed in the Status area of the GS Junior Sequencer software main window. Click OK to continue. 6. You will next perform a ‘dry Pre-wash’, to dry out the instrument’s fluidics system. If the GS Junior Sequencer main window is not open, launch the GS Junior Sequencer application by double-clicking the “GS Junior Sequencer” icon on the desktop. 7. Click the Start button in the Global Action area (Figure 4). This opens the Instrument Procedure Wizard first window: Choose a procedure (Figure 5 A). The Instrument Procedure Wizard has only three windows for the pre-wash procedure.
Figure 4: Start button
8. Select the "Service" Option and click the Next button. This opens the second Instrument Procedure Wizard window: Choose a Service (Figure 5 B). 9. Select the Pre-wash option, and click the Next button. This opens the third Instrument Procedure Wizard window: Start Pre-Wash (Figure 5 C). 10. Click Start to begin the Pre-wash. The Pre-wash will start after a few moments, and proceed to completion (about 30 minutes) without any further user intervention.
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A
B
C
Figure 5: GS Junior Sequencer Instrument Procedure Wizard for the Pre-wash procedure. (A) ‘Choose a procedure’ window with “Service” selected (B) ‘Choose a service’ window with “Pre-Wash” selected (C) ‘Start Pre-Wash’ window
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11. When the dry Pre-wash has completed, click the Exit button in the Global Action area of the GS Junior Sequencer application, then select the check-box that requests the instrument to stop. 12. Observe the Instrument LED: a. It will briefly change from solid yellow to blinking red, b. then change to a cycle of green – yellow – red, c. and then change to a much slower cycle of green – yellow – red – black (LED off). 13. Turn off the instrument’s main power switch (Figure 3, above) only when the LED is in this slow cycle pattern, with the black (LED off) between the red and green. 14. Choose System/Shutdown from the Attendant PC menu bar. a. Wait for the shutdown sequence to scroll by. b. Observe that the Attendant PC turns its power off. 15. Clean the PTP cartridge as follows: a. Open the camera door. b. Carefully remove the used PTP device and the cartridge seal. If you do not already have a used but intact PTP device and cartridge seal available, keep these for future use for pre-wash (before a sequencing Run), maintenance wash (see Section 6.1), or Instrument Test (Section 6.5) procedures. c. Clean the PTP cartridge using a soft bristle brush and Sparkleen solution, being especially careful to remove any bead residues near the inlet and outlet ports or in the seal channel. d. Rinse thoroughly with nanopure water and dry using a lint-free laboratory wipe. 16. Clean the camera face plate as described in the Sequencing Method Manual. 17. Close the camera door. 18. Clean the GS Junior Instrument fluidics area as described in the Sequencing Method Manual. 19. Lower the sipper manifold, and close the instrument cover.
Decontamination of the GS Junior Instrument: If the GS Junior Instrument needs to be decontaminated, the following procedure is suggested:
1. Wearing gloves, clean all surfaces with warm soapy water. 2. Sanitize with a 1:10 bleach solution. 3. After 10 minutes of bleach contact time, rinse surfaces with deionized water.
Such a decontamination (in addition to carrying out the preparation described in this section and a maintenance wash of the fluidics system as described in Section 6.1) is recommended before any service and is required prior to shipping if the GS Junior Instrument is returned to Roche.
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3.3.3 Reactivating the GS Junior Instrument After Storage
1. Turn on the Instrument as described in Section 3.1. 2. Carry out a maintenance wash as described in Section 6.2 to remove any contamination that may have built up in the instrument’s fluidics system during storage. 3. Carry out an Instrument Test as described in Section 6.5 to make sure that the instrument is in proper functioning order.
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4 OVERVIEW OF A SEQUENCING RUN IN THE GS JUNIOR SYSTEM
4.1 Continuous Instrument Operation
The GS Junior Instrument is designed for continuous operation, except when it needs scheduled maintenance (as described in sections 6.4 and 6.6 of this Manual) or when it has been prepared for storage due to a prolonged idle period (as described in Section 3.3). Under typical circumstances, operators do not perform any shutdown procedures.
All Run scripts proceed autonomously, and end with a fluidics maintenance wash, so the instrument is automatically prepared for the next Run. Operators are instructed to remove the sippers, filters and reagents promptly after the Run but to leave the used PicoTiterPlate device (PTP device) in place in the PTP area because the pre-wash, which must be carried out before each sequencing Run, requires that a used PTP device be in place (see the Sequencing Method Manual for details). For these reasons, in normal practice, the removal of the used PTP device and the cleanup of the instrument before the following Run are the responsibility of the next operator.
If the GS Junior Instrument is turned off for any reason, restart it as described in Section 3.1.
4.2 Before You Begin a Sequencing Run
Before starting an experiment, make sure the requirements described in this section are met.
4.2.1 State of the Instrument
As indicated above, the GS Junior Instrument is designed for continuous operation, so it is ready for use as soon as a Run completes. The Status Indicator LED located on the instrument cover shows at a glance what the instrument is doing. Before starting a new Run, therefore, make sure that the previous Run is complete and that the instrument is in the following condition: On the computer screen, the GS Junior Sequencer application window is open. The sipper manifold is in the lower position and the tips of all Sipper Tubes are submerged in their respective reagent containers, in the Reagents cassette. The camera door is closed, with the spent PTP device and seal in place, in the PTP area. The Status Indicator LED is solid green.
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If the instrument is not in this state and the operator cannot verify that it has been properly cleaned, the maintenance wash procedure, described in section 6.2, should be performed before a sequencing Run is started.
4.2.2 What You Should Already Have
Before starting the sequencing Run, make sure that the following are available: Sample: A properly prepared, clonally amplified, bead-immobilized DNA library (Rapid, Paired End, cDNA, or Amplicon library) For more information, see the appropriate library preparation Method Manual and emPCR Method Manual. Run Script and Data Analysis File: As part of the experiment set-up, an appropriate Run script and a Data Analysis scheme must be chosen. For more information, see the Sequencing Method Manual. Non-consumable accessories for the instrument: Table 2 lists the accessories supplied with the GS Junior System. Most of them will be needed for the sequencing Run. One GS Junior Titanium Sequencing Kit and one GS Junior Titanium PicoTiterPlate Kit: These kits contain the reagents and other materials needed for a sequencing Run. For more information on the use of these kits, see the Sequencing Method Manual. Various laboratory equipment and supplies: Various items needed to process the sample and load it on the PTP device For detailed information, see the GS Junior System Tables of Material.
Run scripts and data analysis configuration files are provided by 454 Life Sciences Corp., a Roche company, and come preinstalled on the GS Junior System. Run scripts specify the whole sequence of reagent flows during the Run; while data analysis configuration files set parameters for how the Run data will be processed, yielding final sequencing file outputs.
Run scripts: All Run scripts are written by 454 Life Sciences Corp., a Roche company. Users should never attempt to modify a GS Junior System Run program. For more information on Run scripts, please contact your Roche Representative.
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4.3 Initiating and Monitoring a Sequencing Run
For details on the GS Junior Sequencer and all other software applications, see the 454 Sequencing System Software Manual. For specific instructions on how to set up and carry out a sequencing Run using the GS Junior Sequencer application, see the Sequencing Method Manual.
Sequencing Runs are set up and controlled from GS Junior Sequencer software application, on the Attendant PC. The GS Junior Sequencer application is launched by double-clicking the “GS Junior System” icon on the Attendant PC desktop (Figure 6).
Figure 6: The GS Junior System icon on the Attendant PC desktop
The GS Junior Sequencer application will open, showing a sign-in screen (Figure 7 A). When the application opens, an Instrument check procedure begins, and the status message “Checking Instrument” is displayed. The Instrument check takes approximately 2 minutes to complete. The user can sign in while the Instrument check is running. When the Instrument check has completed, the status message changes to “Available”.
After the Operator has signed in (see the 454 Sequencing System Software Manual for how to set up user accounts), the two main tabs of the GS Junior Sequencer application, Instrument and Data, will appear. After the Instrument check has completed, the Start Button will become available (Figure 7 B). Clicking the Start button opens the Instrument Procedure Wizard through which sequencing Runs are set up and launched.
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A
B
Figure 7: The GS Junior Sequencer application (A) Introduction and Sign-In Form (B) Main window, with the Instrument and Data tabs.
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When a sequencing Run is in progress, it can be monitored from the Instrument tab of the GS Junior Sequencer application. An example is shown on Figure 8.
Figure 8: The GS Junior Sequencer application Instrument tab during a sequencing Run
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5 OVERVIEW OF DATA PROCESSING IN THE GS JUNIOR SYSTEM
For a detailed description of data processing and data analysis in the GS Junior System, see the 454 Sequencing System Software Manual.
The raw output of a sequencing Run is a set of digital images (PNG files) and associated meta-data files which are used to determine the sequence of the DNA library fragments (“reads”). The GS Run Processor application is usually invoked on the GS Junior System Attendant PC as part of the sequencing Run, but can also be run either on the Attendant PC or on a separate computer (‘DataRig’) following the Run. The processing consists of a series of automatic data correction steps that compensate for optical effects and chemical inefficiencies. The compensated reads are then passed through a series of tunable quality filters whereby low quality reads are identified and segregated. Finally, the high quality reads are subjected to an algorithm that converts the signal intensities to individual basecalls and quality scores. The raw data, intermediate processing results, meta-data and metrics data are stored in an archive format. From these composite files, a selection of reports and other artifacts (such as common FASTA files) about the sequencing Run can be generated.
The outputs from one or more sequencing Runs are then further analyzed according to the type of library and the objective of the experiment: Reads from Rapid libraries made from a high molecular weight DNA sample (e.g. shotgun libraries of genomic DNA) can be either assembled into a consensus sequence using the GS De Novo Assembler software, or mapped against a reference sequence using the GS Reference Mapper software. The final consensus sequence is output as a set of FASTA files, with an associated basecall quality score file. The system output also includes: Standard Flowgram Format (SFF) files, files to help visualize the sequence assembly or how the reads were mapped against the reference sequence (ACE format), lists of differences between the consensus reads and the reference sequence(s), if applicable, and various metrics files.
The sequencing output of Rapid libraries made from low molecular weight DNA usually does not require processing beyond raw reads.
Reads from Paired End libraries are normally analyzed in conjunction with those from a shotgun library (made from the same DNA sample), using the GS De Novo Assembler software: Paired End reads are used to order and orient the contigs generated by the shotgun sequencing reads. The output is similar to the one described above, but also contains contig scaffolding information (in AGP format).
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Reads from a cDNA library are used to get information from transcripts in a sample. They can be analyzed either using the GS De Novo Assembler software or the GS Reference Mapper software, depending on whether reference sequence (transcriptomic or genomic) information is available for the sample sequenced. Again, the output is similar to the one described above, but also contains specific information relevant to transcriptomics / cDNA sequencing experiments such as putative exon boundaries, transcripts (isoform) consensus sequences, etc. Reads from Amplicon libraries are analyzed completely differently. A special software application named the GS Amplicon Variant Analyzer aligns the reads against a defined target reference sequence. From the alignment, known and novel Variants are identified, their prevalence quantitated, and then presented both in tabular form and in Variation Frequency Plots. The 454 Sequencing System offers a wide flexibility in the design of Amplicon sequencing experiments, as described in the document entitled 454 Sequencing System Guidelines for Amplicon Experimental Design.
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6 GS JUNIOR INSTRUMENT MAINTENANCE
This section describes certain general procedures you may have to carry out while working with your GS Junior Instrument, either in the course of normal operation or for the maintenance or testing of the instrument. A list of the items covered by the regular service of the instrument performed by a Roche Field Service Representative is also provided. The sub-sections are: Section 6.1: Sipper Tubes, Filters, and Buffer Tray Replacement Section 6.2: Maintenance Wash Section 6.3: Test Sequencing Run with Control DNA Beads Section 6.4: Pump Tubing and Air Filter Replacement Section 6.5: Instrument Test Section 6.6: Roche Preventive Maintenance Section 6.7: Reconfiguring the emPCR Shaker Adapters MV for Older TissueLyser Models
6.1 Sipper Tubes, Filters, and Buffer Tray Replacement
Sipper Tubes and Filters should be replaced between every sequencing Run, maintenance wash, or Instrument Test: new sippers and filters should be installed just prior to the Run and removed promptly thereafter (as soon as convenient and no later than three days after the Run: prompt removal minimizes the risk of biological contaminants growing in your GS Junior Instrument). Also, such replacements should be performed if any Sipper Tube or Filter is known or suspected to be contaminated. (In this case, you may not need to replace all the Sipper Tubes and Filters.)
This procedure can be carried out using the Sipper Tubes and Filters from the GS Junior Titanium Sequencing Kit (Cat No.: 05 996 554 001), the GS Junior Maintenance Wash Kit (see Section 6.2), or using a separate GS Junior Sipper Maintenance Kit (Cat. No. 05 954 070 001). Among other components, these kits contain the following:
Item Sequencing Kit Maint. Wash Kit Sipper Maint. Kit Buffer CB Sipper Tube (long) 1 -- 1 Buffer CB Sipper Filter 1 -- 1 Reagent Sipper Tube (short) 10 10 10 Reagent Sipper Filter 10 10 10 Buffer Tray -- -- 1
At the time of this writing, the GS Junior Titanium Sequencing Kits come with a full complement of Sipper Tubes and Filters. If you are using a Sequencing Kit of the previous format, i.e. prior to Lot Number 93865420, the kit will contain only the Buffer CB Sipper Tube (long) and its Filter. To use these older Sequencing Kits, you must also obtain a GS Junior Sipper Maintenance Kit.
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Certain software versions prior to v2.7 include a Run counter that alerts the user to change Sipper Tubes and Filters after a certain number of Runs. Since the current instructions are to replace all Sipper Tubes and Filters between every Run, you can safely ignore such alerts.
Fluidics contamination: To prevent the contamination of important fluidics components of the GS Junior Instrument and ensure quality results, always wear laboratory gloves while performing these procedures and change gloves as appropriate when gloves become soiled or contaminated during use. Never touch Sipper Tubes with an ungloved hand or a wet gloved hand.
6.1.1 Removing Used Sipper Tubes and Filters
Perform this procedure as soon as convenient and at most within three days following a sequencing Run (see the Sequencing Method Manual), a maintenance wash (see Section 6.2), or an Instrument Test (see Section 6.5).
1. Open the main cover of the GS Junior Instrument and raise the sipper manifold (Figure 9 A). 2. If present, remove the Reagents cassette (Figure 9 B). 3. Remove the Buffer CB (long) Sipper Tube and its Filter (Figure 9 C) and the 10 Reagent (short) Sipper Tubes and Filters from under the manifold by unscrewing the filters (Figure 9 D), and discard them.
The Sipper Tubes and Filters are Luer-Lok-type screws: turning in a clockwise motion tightens them, and turning in a counterclockwise motion loosens them.
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A B
C D
Figure 9: Removing the used Sipper Tubes
(A) Sipper manifold being raised, with the main instrument cover open (B) Removing the Reagents cassette (C) Removing the Buffer CB (long) Sipper Tube and Filter from the sipper manifold; unscrew by holding the filter and turning counterclockwise (D) Removing the Reagent (short) Sipper Tubes and Filters from the sipper manifold; unscrew by holding the filter and turning counterclockwise
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6.1.2 Installing New Sipper Tubes and Filters
Perform this procedure as part of the initiation of a sequencing Run (see the Sequencing Method Manual), a maintenance wash (see Section 6.2), or an Instrument Test (see Section 6.5). Note that this procedure assumes that the sippers and filters used in the previous Run as well as the spent reagents were removed as recommended. If they were not, remove them as described in Section 6.1.1.
The manifold position second from the right on the back row is not used and should not be fitted with a Sipper Tube. Both this position and an air intake position on the left have a pre-installed air filter in them; the air filters in these two positions should not be tampered with. The Sipper Tubes and Filters are Luer-Lok-type screws: turning in a clockwise motion tightens them, and turning in a counterclockwise motion loosens them.
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1. Attach one new Reagent Sipper Tube (short) to a corresponding Filter (Figure 10 A). 2. Holding the Sipper Tube / Filter assembly by the top of the Sipper Tube itself (Figure 10 B), screw it into position on the sipper manifold. Do not overtighten but make sure it is fully seated. 3. Repeat for the other nine Reagent Sipper Tubes and Filters.
Holding the sippers: When installing new Sipper Tubes, make sure to hold the Sipper Tube itself, not the Filter (see Figure 10 B and C). Screwing via the Sipper Tube itself ensures that both the Sipper Tube and the Filter get tightened. Also, only finger-tighten them to avoid breaking the filter; a broken or cracked Filter would prevent suction of the reagent and cause a failure of the next Run.
A
B C
Figure 10: Installation of the Reagent Sipper Tubes (short) and Filters. (A) Assembling the sipper and filter. (B) Proper grip for installing the sipper/filter assembly. (C) Incorrect grip for installing the sipper/filter assembly.
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4. (Skip Steps 4 and 5 if you are preparing for a maintenance wash procedure.) Attach the new Buffer CB Sipper Tube (long) to the corresponding Filter (Figure 11 A). 5. Holding the Sipper Tube / Filter assembly by the top of the Sipper Tube itself (Figure 11 B), screw it into position on the sipper manifold. Do not overtighten but make sure it is fully seated.
Holding the sippers: When installing new Sipper Tubes, make sure to hold the Sipper Tube itself, not the Filter (see Figure 11 B and C). Screwing via the Sipper Tube itself ensures that both the Sipper Tube and the Filter get tightened. Also, only finger-tighten them to avoid breaking the filter; a broken or cracked Filter would prevent suction of the reagent and cause a failure of the next Run.
A
B C
Figure 11: Installation of the Buffer CB Sipper Tube (long) and Filter. (A) Assembling the sipper and filter. (B) Proper grip for installing the sipper/filter assembly. (C) Incorrect grip for installing the sipper/filter assembly.
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6. If using the GS Junior Sipper Maintenance Kit, remove and discard the old Buffer Tray and install the new one in its position, in the front of the Reagents cassette area (Figure 12).
Figure 12: Replacing the Buffer Tray
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6.2 Maintenance Wash
All sequencing Runs end with a maintenance wash that uses a combination of Bleach and Tween reagents. In addition to this, a separate maintenance wash as described in this Section should be performed in the following situations, to prevent contaminants (e.g. bacterial growth or biofilm) from building up in the instrument’s fluidics system: If the instrument has been idle for 30 to 60 days since the last sequencing Run, maintenance wash, or Instrument Test, perform a stand-alone maintenance wash immediately before the next sequencing Run. If the instrument has been put in storage as described in Section 3.3, a maintenance wash should be performed as part of instrument reactivation (see Section 3.3.3). Perform a maintenance wash whenever instructed to do so by a Roche Technical Support Representative, or when you suspect a biological contamination may be present in your instrument’s fluidics system.
Microbial growth in idling instrument: The procedure described in this section should be performed as indicated above. If this procedure is not performed, microbial growth or biofilms may form in the fluidics subsystem of the GS Junior Instrument, causing subsequent sequencing Runs to fail and possibly requiring replacement of parts in the fluidics subsystem.
If you know that the instrument will not be used for an extended period of time (more than 60 days), prepare it for storage as described in section 3.3.
To perform a stand-alone maintenance wash, you must obtain a GS Junior Maintenance Wash Kit (Cat. No.05 889 111 001). The procedure for performing a maintenance wash is similar to a sequencing Run, as described in the Sequencing Method Manual. An abridged form of that procedure is provided below.
PTP device and seal must be present: A used but intact PTP device and seal must be present in the PTP area (in the Camera door of the GS Junior Instrument) to carry out a maintenance wash, or the fluidics will leak. Since the GS Junior Instrument is designed for continuous operation (see Section 4.1), the PTP device and seal from the previous Run are usually present when you prepare for a maintenance wash. If not, install a used but intact PTP device and seal in the PTP area before carrying out the procedure below.
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1. Open the instrument cover and lift the sipper manifold. 2. If present, empty any fluid from the two bottles (Buffer CB and waste), and place them back into position to be used as receptacles for backwash fluid during this procedure. 3. Remove the Reagents cassette if present. 4. Remove and discard any Sipper Tubes that may be present on the sipper manifold. 5. Install the ten short Sipper Tubes from the GS Junior Maintenance Wash Kit, as described in Section 6.1.2.
Note that the maintenance wash procedure does not require a long Sipper Tube for the Buffer CB (front- left) position; only the 10 short Sipper Tubes and Filters.
6. Load the Maintenance Wash Cassette in the GS Junior Instrument (Figure 13 A). 7. Lower the sipper manifold, and close the exterior fluidics door (Figure 13 B).
A B
Figure 13: Installing the Maintenance Wash Cassette and lowering the Sipper Manifold
8. On the Attendant PC, a “Sequencing run complete” message may be displayed in the Status area of the GS Junior Sequencer software main window. Click OK to continue. 9. If the GS Junior Sequencer main window is not open, launch the GS Junior Sequencer application by double-clicking the “GS Junior Sequencer” icon on the desktop.
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10. Click the Start button in the Global Action area (Figure 14). This opens the Instrument Procedure Wizard first window: Choose a procedure (Figure 15 A). The Instrument Procedure Wizard has only three windows for the maintenance wash procedure.
Figure 14: Start button
11. Select the "Service" Option and click the Next button. This opens the second Instrument Procedure Wizard window: Choose a Service (Figure 15 B). 12. Select the Maintenance wash option, and click the Next button. This opens the third Instrument Procedure Wizard window: Start Maintenance Wash (Figure 15 C). 13. Click Start to begin the maintenance wash. The maintenance wash will start after a few moments, and proceed to completion (about 60 minutes) without any further user intervention. 14. As soon as possible and no more than three days after the end of the maintenance wash, do the following: a. Open the instrument cover and lift the sipper manifold. b. Empty the back-flushed fluid from the two bottles and keep one bottle for future use as waste receptacle. c. Remove and discard the Maintenance Wash Cassette and all the Sipper Tubes and Filters attached to the sipper manifold, as described in Section 6.1.1.
Sipper Tube replacement: Replacement of the Sipper Tubes and Filters after a maintenance wash is critically important. If the Sipper Tubes and Filters were not replaced, residual bleach in the filters would interfere with the following sequencing Run, possibly resulting in a Run failure. Fresh Sipper Tubes and Filters for the next sequencing Run will be provided in the GS Junior Titanium Sequencing Kit.
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A
B
C
Figure 15: GS Junior Sequencer Instrument Procedure Wizard for a maintenance wash. (A) ‘Choose a procedure’ window with “Service” selected. (B) ‘Choose a service’ window with “Maintenance Wash” selected. (C) ‘Start Maintenance Wash’ window.
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6.3 Test Sequencing Run with Control DNA Beads
Under certain circumstances, your GS Junior Service Representative may ask you to perform a sequencing Run with Control Beads from the GS Junior Titanium Control Bead Kit (Cat. No. 05 996 643 001) to verify that your GS Junior Instrument is performing to specifications. This test Run uses Control Beads XLTF as sample, along with a GS Junior Titanium Sequencing Kit and a GS Junior Titanium PicoTiterPlate Kit.
The Sequencing Method Manual describes in detail how to perform a normal sequencing Run. Except for the use of the GS Junior Titanium Control Bead Kit, the procedure to perform a test sequencing Run is identical to a normal sequencing Run, as described in the Sequencing Method Manual: simply substitute the instruction of Section 3.2.3.1: Prepare the DNA Beads in the Sequencing Method Manual, with the following:
1. Vortex the two tubes of Control Beads XLTF to obtain homogeneous suspensions, and transfer 100 μl (50 μl from each tube) of bead suspension into a 1.7 ml tube. 2. Add 500 μl of BB2 to the DNA Beads, vortex gently, and incubate for 20 minutes at room temperature on a lab rotator.
During this 20 minute incubation, prepare the Packing Beads, Enzyme Beads and PPiase Beads.
Guidance for analysis of sequencing results using GS Junior Control DNA Beads is available from your GS Junior System Technical Support Representative.
6.4 Pump Tubing and Air Filter Replacement
This procedure requires a GS Junior Preventive Maintenance Kit (Cat. No. 05 898 765 001), which contains the following: 2 Pieces of Pump Tubing 1 Air Filter 2 BDD Adapters A 2 BDD Adapters B
The BDD Centrifuge Adapters (A and B) are not used in the procedure below. They are provided in the GS Junior Preventive Maintenance Kit in case replacement is needed, e.g. if any Adapters are torn or otherwise damaged, or if they have been misplaced.
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The Pump Tubing replacement procedure should be carried out when a warning message appears (Figure 16) to remind you that Pump Tubing replacement is due. This comes from a Run counter that resides in the GS Junior Instrument and which will need to be reset after the Pump Tubing is replaced (as described below). It is good practice to replace the instrument’s Air Filter at the same time, though no warning reminder is provided for Air Filter replacement.
Figure 16: Message indicating that the Pump Tubings need to be replaced
Fluidics contamination: To prevent the contamination of important fluidics components of the GS Junior Instrument and ensure quality results, always wear laboratory gloves while performing these procedures and change gloves as appropriate when gloves become soiled or contaminated during use. Never touch the Pump Tubing with an ungloved hand or a wet gloved hand.
6.4.1 Replacing the Pump Tubing
1. Open the instrument cover to access the pumps area located on top of the sipper manifold. 2. Choose one side to start with (left pump in this example) and pull out the fittings from the mounting clips (Figure 17 A). 3. Disconnect the fittings from both ends of the Pump Tubing (Figure 17 B). 4. Pull gently to unsnap the tubing stop of the long (top) end of the Pump Tubing from the pump housing, then manually rotate the pump rotor and pull on the Pump Tubing in the direction of rotation to remove it completely from inside the pump housing (Figure 17 C).
Apply the force to the pump rotor, not the motor spindle when performing this step.
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A B
C
Figure 17: Removing the pump tubing (A) Pull the fittings out of the mounting clips (B) Disconnect the fittings from the Pump Tubing (C) Pull the Pump Tubing out of the pump housing
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5. Before inserting the new Pump Tubing, rotate the pump rotor to provide clearance at the bottom entry point into the pump housing, as shown in Figure 18.
Figure 18: Rotate the pump rotor to provide clearance at the bottom entry point into the pump housing, as shown in the left panel.
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6. Insert the stop of the short end of the Pump Tubing into the bottom position of the pump housing (Figure 19 A).
The Pump Tubing must be mounted in the proper orientation, short end at the bottom, for proper flow direction.
7. Manually rotate the pump rotor and guide the Pump Tubing into the housing in the direction of rotation (Figure 19 B). 8. Gently push the long end (top) Pump Tubing stop to snap it into the pump housing (Figure 19 C). 9. Reconnect both ends of the Pump Tubing to their fittings, and snap them back into the mounting clips (Figure 19 D). 10. Tuck away the long (top) end of the Pump Tubing so it does not protrude above the sipper manifold cover.
A B
C D
Figure 19: Installing new Pump Tubing (A) Inserting the new Pump Tubing into the pump housing (B) Guiding the Pump Tubing into the pump housing (C) Pushing the long end (top) Pump Tubing stop into the pump housing (D) Snapping the Pump Tubing connector fittings into the mounting clips.
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11. Repeat steps 2 through 10 for the Pump Tubing of the other pump. 12. To reset the Pump Tubing replacement counter, do the following: a. Click the Start button in the Global Action area (Figure 20). This opens the Instrument Procedure Wizard first window: Choose a procedure (Figure 21 A). b. Select the "Service" Option and click the Next button. This opens the next Instrument Procedure window: Choose a Service (Figure 21 B). c. Select the Custom script option, and click the Next button. This opens the next Instrument Procedure window: Select an Instrument Run script (Figure 21 C). d. Click the folder icon to the right of the Instrument script field. This opens the Choose Instrument Run Script File dialog (Figure 21 D). e. Navigate to the Service folder. The default location of this folder is: /opt/454/apssgsSequencer/runScripts/Service. f. Locate and launch the script named Record_Tubing_Replacement.scs by selecting it and clicking the Choose button. This action returns the focus to the Select an Instrument Run script window, with the selected script now in the Instrument script field. g. Click the Next button. This opens the next Instrument Procedure window describing the selected script (Figure 21 E). h. Click the Start button. The Instrument Procedure Wizard will close and the output of the script will appear in the Instrument tab of the GS Junior Sequencer application (Figure 21 F). i. When the script has completed, the OK button will become available. Click the OK button. 13. Return to the ‘Pump tubing need to be replaced’ Warning message (Figure 16) and click on Recheck to initiate an instrument Self-Test. 14. When the Self-Test is complete (and does not return an error), perform an Instrument Test as described in Section 6.5.
Figure 20: Start button
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A B
C D
E F
Figure 21: Resetting the Pump Tubing replacement counter. (A) ‘Choose a procedure’ window with “Service” selected; (B) ‘Choose a service’ window with “Custom script” selected; (C) ‘Select an Instrument Run Script’ window showing the folder icon, to the right of the entry field; (D) Choose Instrument Run Script File dialog showing the contents of the “Service” folder and the “Record_Tubing_Replacement.scs” script selected; (E) ‘Special Command Review’ window providing a brief description of the selected script and the available Start button; (F) Portion of the GS Junior Sequencer window showing the script output and the OK button.
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6.4.2 Replacing the Air Filter
1. Remove the Air Filter cover (Figure 22 A). 2. Remove the System Air Filter from its cover. 3. Place the new System Air Filter into the cover (Figure 22 B). 4. Place the filter cover back into position.
A B
Figure 22: Replacing the System Air Filter
6.5 Instrument Test
This procedure consists of a pump calibration test (as is carried out behind the scenes at the beginning of each sequencing Run), followed by a verification of the proper functioning of various components of the GS Junior Instrument including the PTP device heater, the reagent chiller, and the CCD camera chiller. The test also verifies the flow rate through each valve and other script-controlled Run parameters. It returns a simple Pass or Fail result with instructions to contact your Roche Technical Service Representative in case of a Failed test.
The procedure requires a GS Junior Titanium Sequencing Buffers Kit (Cat. No.: 06 627 846 001) containing the components listed in Table 4. The procedure should be carried out in the following circumstances: When the pump tubing replacement procedure (Section 6.4) has been carried out. After a software update on the GS Junior Instrument and/or its Attendant PC. As part of the procedure to reactivate an instrument that is being taken out of long-term storage (see Section 3.3.3). Whenever instructed to do so by a Roche Technical Support Representative, or when you want to verify that your instrument is in proper functioning order.
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Contents Item Description 1 Bottle, 1000 ml Pre-wash Buffer 1 Bottle, 1100 ml Buffer CB* 1 Cassette, 10 tubes Pre-wash Cassette 1 Sipper Tube Buffer CB Sipper Tube (long) 1 Filter Buffer CB Sipper Filter 10 Sipper Tubes Reagent Sipper Tube (short) 10 Filters Reagent Sipper Tube Filter
Table 4: Components of the GS Junior Titanium Sequencing Buffers Kit (Cat. No.: 06 627 846 001).
*Note that the Buffer CB is not used in this procedure.
The bottle of Buffer CB is not used in this procedure.
PTP device and seal must be present: A used but intact PTP device and seal must be present in the PTP area (in the Camera door of the GS Junior Instrument) to carry out the Instrument Test, or the fluidics will leak. Since the GS Junior Instrument is designed for continuous operation (see Section 4.1), the PTP device and seal from the previous Run are usually present when you prepare for an Instrument Test. If not, install a used but intact PTP device and seal in the PTP area before carrying out the procedure below.
1. Open the instrument cover and lift the sipper manifold. 2. If present, empty any fluid from the two bottles (Buffer CB and waste), but keep one bottle to be used as waste receptacle. 3. If present, remove and discard the Reagents cassette and any Sipper Tubes and Filters that may be attached to the sipper manifold, as described in Section 6.1.1. 4. Install the ten short Sipper Tubes and Filters as well as the single long Sipper Tube and its Filter to their proper positions on the sipper manifold, as described in Section 6.1.2. 5. Change gloves to avoid contaminating other components. 6. Pull the Pre-wash cassette from its bag and rinse the ten tubes of the Pre-wash cassette twice with nanopure water.
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7. Fill the ten tubes with Pre-wash Buffer up to approx. 1 cm from the top of the tubes (Figure 23).
Figure 23: Filling the Pre-wash cassette
8. Load the Pre-wash cassette in the GS Junior Instrument, and place the bottle with the remaining Pre-wash Buffer on the front-left position and the waste bottle on the front-right position of the instrument (Figure 24 A). 9. Lower the sipper manifold, and close the exterior fluidics door (Figure 24 B).
A B
Figure 24: Installing the Pre-wash cassette and lowering the sipper manifold
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10. On the Attendant PC, a “Sequencing run complete” message may be displayed in the Status area of the GS Junior Sequencer software main window. Click OK to continue. 11. If the GS Junior Sequencer main window is not open, launch the GS Junior Sequencer application by double-clicking the “GS Junior Sequencer” icon on the desktop. 12. Click the Start button in the Global Action area (Figure 25). This opens the Instrument Procedure Wizard first window: Choose a procedure (Figure 26 A). The Instrument Procedure Wizard has only three windows for the Instrument Test procedure.
Figure 25: Start button
13. Select the "Service" Option and click the Next button. This opens the second Instrument Procedure Wizard window: Choose a Service (Figure 26 B). 14. Select the Instrument Test option, and click the Next button. This opens the third Instrument Procedure Wizard window: Start Instrument Test (Figure 26 C). 15. Click Start to begin the Instrument Test. The Instrument Test will start after a few moments, and proceed to completion (about 60 minutes) without any further user intervention.
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A
B
C
Figure 26: GS Junior Sequencer Instrument Procedure Wizard for Instrument Tests. (A) ‘Choose a procedure’ window with “Service” selected; (B) ‘Choose a service’ window with “Instrument Test” selected; (C) ‘Start Instrument Test’ window.
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16. When the Instrument Test has completed, a message window indicating whether your instrument passed or failed the test will open (Figure 27). a. If the instrument passed the test, it is ready for any further operation. b. If the instrument failed the test, please call your Roche Technical Service Representative as instructed.
A B
Figure 27: Messages indicting the result of an Instrument Test procedure: (A) Passed; (B) Failed
17. As soon as possible and no more than three days after the end of the Instrument Test, do the following: a. Open the instrument cover and lift the sipper manifold. b. Empty any remaining fluid from the two bottles (Pre-wash Buffer and waste); discard the waste bottle but keep the bottle of Pre-wash Buffer for future use as waste receptacle. c. Remove and discard the Pre-wash cassette and all Sipper Tubes and Filters attached to the sipper manifold, as described in Section 6.1.1.
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6.6 Roche Preventive Maintenance
Roche preventive maintenance typically occurs annually or after 100 Runs, whichever occurs first. Roche preventive maintenance actions may include, but are not limited to: Cleaning the GS Junior Instrument Pump tubing and rotor replacement Sequencer tubing lines and fitting verification/replacement Sipper assembly and valve manifold connection verification/adjustment Maintenance wash (if necessary)
This preventive maintenance may only be performed by a Roche Field Service Engineer. Contact your local Roche Service Representative for more details on national-specific service contracts.
6.7 Reconfiguring the emPCR Shaker Adapters MV for Older TissueLyser Models
The GS Junior System supports the use of the GS FLX Titanium MV emPCR Kits (Lib-L or Lib-A) to amplify multiple libraries at the same time (see the corresponding “Multiple Prep” emPCR amplification Method Manuals). To use those kits, a pair of Shaker Adapters MV (Cat. No.: 05 618 487 001) is required. These Adapters come pre- assembled for use with the TissueLyser II (Qiagen), with the attachment cylinders in the central position. In order to prevent the emulsion oil tube caps from interfering with the safety shield on previous TissueLyser models, the Shaker Adapters MV must be reconfigured with the attachment cylinders in the side (lower) position, as described below and on Figure 28.
For each Shaker Adapter MV:
1. Remove the black thumb screw and the aluminum bracket from the white tube holder. 2. Using a 2.5 mm hex wrench, remove the four flat head screws that secure the white tube holder to the attachment plate. This will expose the two attachment plate’s screws. 3. Using a 4 mm hex wrench, remove the two cap head screws that secure the attachment plate to the attachment cylinder. 4. Using a 1.5 mm hex wrench, install two anti-rotation screws from the accessory pack into the outside pre-tapped holes on the lip at either end of the attachment cylinder. 5. Re-assemble the attachment cylinder into the off-center position of the attachment plate (two cap head screws). Make sure to orient it such that the cylinder’s anti-rotation screws are toward the center of the plate. 6. Re-assemble the tube holder with the attachment plate (4 flat head screws). Make sure to assemble the two Shaker Adapters MV in opposite orientation from one another such that when they are mounted into the TissueLyser, they will both have the tube caps pointing outward (see Figure 28, bottom-left).
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Figure 28: Re-configuration of the Shaker Adapters MV, for TissueLyser models prior to the TissueLyser II.
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Published by 454 Life Sciences Corp. A Roche Company Branford, CT 06405 USA
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