DOCSLIB.ORG

454 Sequencing System Guidelines for Amplicon Experimental Design

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
454 Sequencing System Guidelines for Amplicon Experimental Design 454 Sequencing System Guidelines for Amplicon Experimental Design June 2013 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. Guidelines for Amplicon Experimental Design 454 Sequencing System 454 Sequencing System Guidelines for Amplicon Experimental Design Table of Contents 1 General Guidelines for Amplicon Sequencing Experimental Design ...................... 5 1.1 Basic Considerations ................................................................................................................................. 6 1.2 Depth of Coverage ..................................................................................................................................... 6 1.3 Multiplexing ................................................................................................................................................. 7 1.3.1 Multiplex PCR ........................................................................................................................................................................ 7 1.3.2 Multiplex Identifiers (MIDs) ............................................................................................................................................. 7 1.4 Bidirectional vs. Unidirectional Sequencing ........................................................................................ 8 1.5 Quantitative Results in Amplicon Sequencing ..................................................................................... 8 2 Five Types of Amplicon Sequencing Experimental Design ........................................ 9 3 Design 1: Basic Amplicon Sequencing ........................................................................ 11 3.1 Library preparation................................................................................................................................... 12 3.1.1 Kit and Method .................................................................................................................................................................. 12 3.1.2 Multiplexing......................................................................................................................................................................... 13 3.1.3 Fusion Primer Design ...................................................................................................................................................... 14 3.1.4 Amplicon Length Considerations ............................................................................................................................... 15 3.1.5 Fusion Primer Ordering .................................................................................................................................................. 15 3.2 emPCR Amplification ............................................................................................................................... 16 3.2.1 Kits and Methods .............................................................................................................................................................. 16 3.2.2 Library Pooling ................................................................................................................................................................... 16 3.3 Sequencing ................................................................................................................................................ 17 3.3.1 Kit and Method .................................................................................................................................................................. 17 3.3.2 Pooling ................................................................................................................................................................................... 17 3.4 Data Processing........................................................................................................................................ 18 3.5 Data Analysis............................................................................................................................................. 18 4 Design 2: Universal Tailed Amplicon Sequencing ..................................................... 19 4.1 Library preparation................................................................................................................................... 20 4.1.1 Kit and Method .................................................................................................................................................................. 20 4.1.2 Multiplexing......................................................................................................................................................................... 21 4.1.3 Fusion Primer Design ...................................................................................................................................................... 22 4.1.3.1 Adding the Universal Tails ................................................................................................................................ 22 4.1.3.2 Targeting the Universal Tails ............................................................................................................................ 22 4.1.4 Amplicon Length Considerations ............................................................................................................................... 24 4.1.5 Fusion Primer Ordering .................................................................................................................................................. 24 June 2013 3 Guidelines for Amplicon Experimental Design 454 Sequencing System 4.2 emPCR Amplification ............................................................................................................................... 24 4.3 Sequencing ................................................................................................................................................ 24 4.4 Data Processing........................................................................................................................................ 24 4.5 Data Analysis............................................................................................................................................. 24 5 Design 3: Ligated Adaptors Amplicon Sequencing ................................................... 25 5.1 Library Preparation .................................................................................................................................. 26 5.2 emPCR Amplification ............................................................................................................................... 27 5.3 Sequencing ................................................................................................................................................ 27 5.4 Data Processing........................................................................................................................................ 27 5.5 Data Analysis............................................................................................................................................. 27 6 Design 4: Long Range PCR Amplicon Sequencing ................................................... 28 6.1 Library Preparation .................................................................................................................................. 29 6.2 emPCR Amplification ............................................................................................................................... 29 6.3 Sequencing ................................................................................................................................................ 29 6.4 Data Processing........................................................................................................................................ 29 6.5 Data Analysis............................................................................................................................................. 29 7 Design 5: One-Way Reads Amplicon Sequencing..................................................... 31 7.1 Library preparation................................................................................................................................... 33 7.1.1 Kit and Method .................................................................................................................................................................. 33 7.1.2 Multiplexing......................................................................................................................................................................... 34 7.1.3 Fusion Primer Design ...................................................................................................................................................... 34 7.1.4 Amplicon Length Considerations ............................................................................................................................... 35 7.1.5 Fusion Primer Ordering .................................................................................................................................................. 35 7.2 emPCR Amplification ..............................................................................................................................
Load more

Recommended publications
  • Grant Application Resources for Visium Spatial Gene Expression
    10x Genomics | Visium | Spatial Gene Expression Grant Application Resources Grant application resources for Visium Spatial Gene Expression Summary statement Visium Spatial Gene Expression from 10x Genomics is a novel assay that combines histology with spatially resolved whole transcriptome gene expression profiling to localize and quantify gene expression in the tissue context. It is based on the Spatial Transcriptomics methodology (1). The assay has been well adopted, being utilized in almost 40 peer-reviewed publications and over 50 pre-prints. Currently, Visium Spatial Gene Expression is compatible with fresh frozen tissue sections from any species. This assay utilizes poly(A) capture and novel spatial barcoding technology for library preparation. 10x Genomics also offers a Visium Spatial Gene Expression assay compatible with human and mouse formalin-fixed paraffin-embedded (FFPE) tissue sections. This assay utilizes RNA-templated ligation of pairs of gene target probes for highly specific and sensitive detection of the whole transcriptome. Both assays leverage the same suite of analysis tools and pipelines (e.g., Space Ranger, Loupe Browser) to process and visualize Visium Spatial data. Additionally, researchers have access to 10x Genomics technical experts who can provide support through scientific and technical consultations, workflow optimization, and methodology troubleshooting. Overview The ability to detect and count transcripts by sequencing (RNA-seq) has led to significant advances in our understanding of biology (2), as well as the development of clinical applications. However, traditional RNA-seq suffers from the loss of spatial information. Researchers typically extract RNA from tissue and sequence it in bulk. Data regarding the type of cells expressing a given transcript, the location of these cells within the tissue, and co-expression of transcripts in the tissue geography are lost by this bulk preparation of RNA.
    [Show full text]
  • MYC-Containing Amplicons in Acute Myeloid Leukemia: Genomic Structures, Evolution, and Transcriptional Consequences
    Leukemia (2018) 32:2152–2166 https://doi.org/10.1038/s41375-018-0033-0 ARTICLE Acute myeloid leukemia Corrected: Correction MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences 1 1 2 2 1 Alberto L’Abbate ● Doron Tolomeo ● Ingrid Cifola ● Marco Severgnini ● Antonella Turchiano ● 3 3 1 1 1 Bartolomeo Augello ● Gabriella Squeo ● Pietro D’Addabbo ● Debora Traversa ● Giulia Daniele ● 1 1 3 3 4 Angelo Lonoce ● Mariella Pafundi ● Massimo Carella ● Orazio Palumbo ● Anna Dolnik ● 5 5 6 2 7 Dominique Muehlematter ● Jacqueline Schoumans ● Nadine Van Roy ● Gianluca De Bellis ● Giovanni Martinelli ● 3 4 8 1 Giuseppe Merla ● Lars Bullinger ● Claudia Haferlach ● Clelia Tiziana Storlazzi Received: 4 August 2017 / Revised: 27 October 2017 / Accepted: 13 November 2017 / Published online: 22 February 2018 © The Author(s) 2018. This article is published with open access Abstract Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This 1234567890();,: revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres.
    [Show full text]
  • Make Illumina Part of Your DNA. the Most Comprehensive Set of DNA Analysis Tools Available
    Make Illumina part of your DNA. The most comprehensive set of DNA analysis tools available. Illumina’s portfolio of DNA analysis products delivers industry-leading data quality at the lowest cost per study. Accelerate discovery by looking at DNA from every angle. 4 SNP genotyping — Achieve significance with the industry’s best genomic coverage and data quality. 4 CNV analysis — Obtain the most comprehensive access to known and novel CNV regions. 4 DNA sequencing — Sequence virtually anything at a fraction of the cost and time. 4 DNA methylation — Get single CpG site resolution with high- multiplex standard or custom methylation panels. 4 ChIP-Seq — Obtain genome-wide maps of DNA-protein interactions with unprecedented resolution, quality and cost. Make us part of your DNA. Join the growing Illumina Community. Find out how to make Illumina part of your DNA: www.illumina.com/dna?gr Random Shear BAC Library Services Got Gaps? Eliminate gaps in genomic libraries with Lucigen’s exclusive Random Shear BAC Library technical expertise. Resulting in: • Complete genome sequence with as low as 5x coverage • Elimination of restriction site bias found in conventional libraries by utilizing random shearing of DNA into BAC-size fragments • Vast reduction of deleted and rearranged sequences by cloning in our Transcription-Free BAC vectors • Greatly reduced finishing costs. Detailed information on the benefits of Random Shear BAC Libraries for genome projects is available on-line: www.lucigen.com/BAC.pdf Closing centromeric gaps with a Random Shear BAC Library 80 M Notl cut Random Shear BAC clones M 60 40 Centromere 1 20 (~9 Mb) kb No.
    [Show full text]
  • Molecular Thermodynamics of the Stability of Natural, Sugar
    MOLECULAR THERMODYNAMICS OF THE STABILITY OF NATURAL, SUGAR AND BASE-MODIFIED DNA DUPLEXES AND ITS APPLICATION TO THE DESIGN OF PROBES AND PRIMERS FOR SENSITIVE DETECTION OF SOMATIC POINT MUTATIONS by Curtis Hughesman B.A.Sc., The University of Calgary, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2012 © Curtis Hughesman, 2012 Abstract Cancer is characterized as a genetic disease associated with acquired somatic mutations, a majority of which consist of only a single base change and are commonly referred to as somatic point mutations (SPM). Real-time quantitative polymerase-chain reaction (qPCR) techniques using allele specific (AS) probes or primers are widely used in genotyping assays to detect commonly known single nucleotide polymorphisms (SNP), and also have the potential to detect SPMs, provided the required analytical sensitivity and specificity can be realized. One strategy to establish the necessary performance is to introduce nucleotide analogs such as Locked Nucleic Acids (LNAs) into AS probes or primers; however the successful design requires a fundamental understanding of both the thermodynamics and kinetics of LNA-DNA heteroduplexes. Melting thermodynamic studies of DNA duplexes and LNA-DNA heteroduplexes were therefore carried out using both ultraviolet (UV) spectroscopy and differential scanning calorimetry (DSC) to quantify the o o thermodynamics (ΔH , ΔS , ΔCp and Tm) associated with the helix-to-coil transition. Data collected on DNA duplexes and DNA-LNA heteroduplexes were used to introduce improvements in the “unified” nearest-neighbor model, and for the development of a new model, referred to as the Single Base Thermodynamic (SBT) model that accurately predicts the Tm for the melting of LNA-DNA heteroduplexes.
    [Show full text]
  • Short Tandem Repeat Typing on the 454 Platform
    SHORT TANDEM REPEAT TYPING ON THE 454 PLATFORM: STRATEGIES AND CONSIDERATIONS FOR TARGETED SEQUENCING OF COMMON FORENSIC MARKERS Melissa Scheible1,2,*, Odile Loreille1,2, Rebecca Just1,2 and Jodi Irwin1,2,3 1American Registry of Pathology 2Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner System 3Present affiliation: Federal Bureau of Investigation, Quantico The past several years have seen a dramatic advance in the methods, chemistries and detection platforms available for DNA sequence data generation. Next generation sequencing (NGS) technologies, which produce large volumes of sequence data at extremely low cost relative to current platforms, are being broadly applied to various questions in medical genetics, evolutionary biology, molecular anthropology, phylogeny, epidemiology and metagenomics. For many of these applications, NGS is being used to produce sequence data covering thousands of loci, or even entire organismal genomes in a single sequencing run. Given this capacity, it’s not difficult to envision the potential implications of this technology for criminalistics, missing persons and disaster victim identification purposes. Historically, the recovery of large numbers of forensic markers in a single assay has been restricted by both the technical limitations of current, established capillary based sequencing genotyping technologies, as well as the quality and quantity of DNA originating from the damaged and degraded specimens regularly encountered in forensic casework. These limitations don’t apply in quite the same way to NGS, however. As a result, the simultaneous recovery of the standard autosomal DNA, mitochondrial DNA, and X and Y-chromosomal markers regularly assayed in forensic genetics, along with additional markers of interest, may be possible with these new technologies [1].
    [Show full text]
  • Amplicons in Breast Cancers Analyzed by Multiplex Ligation-Dependent
    Human Pathology (2019) 85,33–43 www.elsevier.com/locate/humpath Original contribution Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization☆,☆☆ Akishi Ooi MD, PhD a,b,⁎, Masafumi Inokuchi MD, PhD c, Shin-ichi Horike PhD d, Hiroko Kawashima MD, PhD e, Satoko Ishikawa MD, PhD c, Hiroko Ikeda MD, PhD b, Ritsuko Nakamura MD, PhD a, Takeru Oyama MD, PhD a, Yoh Dobashi MD, PhD f aDepartment of Molecular and Cellular Pathology, Kanazawa University, Ishikawa 920-8641, Japan bPathology Section, University Hospital, Kanazawa University, Ishikawa 920-8641, Japan cDepartment of Breast Oncology, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-8641, Japan dAdvanced Science Research Center, Institute for Gene Research, Kanazawa University, Ishikawa 920-8641, Japan eSection of Breast Oncology, University Hospital, Kanazawa University, Ishikawa 920-8641, Japan fDepartment of Pathology, Saitama Medical Center, Jichi Medical University, Saitama, 330-8503, Japan Received 16 August 2018; revised 12 October 2018; accepted 18 October 2018 Keywords: Summary Gene amplification is a common event in breast cancer, and identifies actual and potential targets of Breast cancer; molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are Gene amplification; reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded in- FISH; vasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a MLPA; total of 906 gene loci judged as ‘gain’ or ‘amplified’ was further confirmed to have been amplified based on Co-amplication fluorescence in situ hybridization (FISH).
    [Show full text]
  • Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci
    Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on August 30, 2012 - Published by Cold Spring Harbor Laboratory Press Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci Katherine E. Varley and Robi D. Mitra Cold Spring Harb Protoc 2009; doi: 10.1101/pdb.prot5252 Email Alerting Receive free email alerts when new articles cite this article - click here. Service Subject Browse articles on similar topics from Cold Spring Harbor Protocols. Categories Amplification of DNA by PCR (51 articles) Bioinformatics/Genomics, general (130 articles) DNA Sequencing (52 articles) Genetic Variation (69 articles) Genetics, general (316 articles) Genome Analysis (97 articles) Genomic DNA (70 articles) High-Throughput Analysis, general (94 articles) Molecular Biology, general (977 articles) Polymerase Chain Reaction (PCR) (61 articles) Polymerase Chain Reaction (PCR), general (136 articles) To subscribe to Cold Spring Harbor Protocols go to: http://cshprotocols.cshlp.org/subscriptions Downloaded from http://cshprotocols.cshlp.org/ at WASHINGTON UNIV SCHOFMED on August 30, 2012 - Published by Cold Spring Harbor Laboratory Press Protocol Nested Patch PCR for Highly Multiplexed Amplification of Genomic Loci Katherine E. Varley and Robi D. Mitra1 Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108, USA INTRODUCTION Nested Patch polymerase chain reaction (PCR) amplifies a large number (greater than 90) of targeted loci from genomic DNA simultaneously in the same reaction. These amplified loci can then be sequenced on a second-generation sequencing machine to detect single nucleotide polymorphisms (SNPs) and mutations. The reaction is highly specific: 90% of sequencing reads match targeted loci. Nested Patch PCR can be performed on many samples in parallel, and by using sample-specific DNA barcodes, these can be pooled and sequenced in a single reaction.
    [Show full text]
  • An Optimization and Common Troubleshooting Solving in Polymerase Chain Reaction Technique Shaden M
    Sys Rev Pharm 2020; 11(2): 427 436 A multifaceted review journal in the field of pharmacy E-ISSN 0976-2779 P-ISSN 0975-8453 An Optimization and Common Troubleshooting Solving in Polymerase Chain Reaction Technique Shaden M. H. Mubarak1; Dhafer A. F. Al-Koofee2*; Ohood A Radhi3; Jawad Mohammed Ismael4; Zubaida Falih Al-Zubaidi5 1,2,5Dept. of Clinical Laboratory Science/Faculty of Pharmacy/University of Kufa 3 Faculty of Nursing/ University of Kufa 4 Dept. of Biochemistry/Faculty of Medicine / University of Kufa P.O. Box (21), Najaf Governorate, Iraq *Corresponding Author E-mail: [email protected] Article History: Submitted: 18.11.2019 Revised: 28.01.2020 Accepted: 29.02.2020 ABSTRACT Many genetic researches now relies on the study of variants in genetic Correspondence: material through different, diverse of universal Polymerase Chain Dhafer A.F.Al-Koofee Reaction (PCR) methods. In that context, we are putting a highlighted Department of Clinical Laboratory Science on the most important fundamental aspects of PCR technology, which Faculty of Pharmacy help researchers to clarify and reduce the majority problems and University of Kufa difficulties may face them in the laboratory work of any kind of PCR E-mail: [email protected] technology in general. DOI: 10.5530/srp.2020.2.63 Keywords: PCR optimization; primer design; melting temperature; PCR troubleshooting; GC-high; DMSO; genetics. @Advanced Scientific Research. All rights reserved INTRODUCTION should be neither high nor low to avoiding produce 1. Rules of PCR primer design insufficient primer-template hybridization and low PCR From a common molecular biology laboratory technique product in high temperature, and non-specific products is Polymerase Chain Reaction (PCR) that utilized to build caused by a high number of base pair mismatches in low up enough many copies of a definite section of DNA for temperature.
    [Show full text]
  • Qiaseq 1-Step Amplicon Library Preparation Handbook 08/2021
    August 2021 QIAseq® 1-Step Amplicon Library Preparation Handbook For the construction of Illumina®-compatible libraries from multiplexed PCR amplicons Sample to Insight__ Contents Kit Contents ............................................................................................................... 4 Shipping and Storage ................................................................................................. 5 Intended Use .............................................................................................................. 6 Safety Information ....................................................................................................... 6 Quality Control ........................................................................................................... 6 Introduction ................................................................................................................ 7 Principle and procedure .................................................................................... 7 NGS adapter and index technologies ................................................................. 9 Starting materials ........................................................................................... 11 Equipment and Reagents to Be Supplied by User .......................................................... 12 Important Notes ........................................................................................................ 13 Recommended library quantification method ....................................................
    [Show full text]
  • Primeval: Optimization and Screening of Multiplex Oligonucleotide Assays Rick Conzemius *, Michaela Hendling, Stephan Pabinger & Ivan Barišić
    www.nature.com/scientificreports OPEN PRIMEval: Optimization and screening of multiplex oligonucleotide assays Rick Conzemius *, Michaela Hendling, Stephan Pabinger & Ivan Barišić The development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecifc hybridization events, the generation of unspecifc by-products, primer depletion, and thus lower amplifcation efciencies. We have developed a software workfow with three underlying algorithms that difer in their use case and specifcity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self- dimers, cross-dimers and hairpins at diferent experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at. Te specifcity of oligonucleotides is essential in nucleic acid techniques such as DNA amplifcation and detection technologies1,2. While classical microbiological methods are commonly used for the identifcation and charac- terization of pathogens, cultivation-independent genetic methods such as (real-time) polymerase chain reaction (PCR) and DNA microarrays are on the rise3. Tese methods are only cost-efective if they are highly multiplexed, which is challenging due to primer dimer formation, the formation of unwanted by-products,
    [Show full text]
  • Multiplexed, High-Throughput Amplicon Diversity Profiling Using Unique Molecular Identifiers
    bioRxiv preprint doi: https://doi.org/10.1101/538587; this version posted February 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Title: MAUI-seq: Multiplexed, high-throughput amplicon diversity profiling using unique molecular identifiers Authors: 1* 2* 1 2 Bryden Fields ​ and Sara Moeskjær ,​ Ville-Petri Friman ,​ Stig U. Andersen ,​ and J. Peter W. ​ ​ ​ ​ Young1 ​ *: These authors contributed equally to the work. Author affiliations: 1 Department​ of Biology, University of York, York, United Kingdom 2 Department​ of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark Authors for correspondence: Peter Young, [email protected] Stig U. Andersen, [email protected] Keywords: ​ Amplification bias, Environmental amplicon diversity, High-throughput amplicon sequencing, Rhizobium, Unique molecular identifier 1 bioRxiv preprint doi: https://doi.org/10.1101/538587; this version posted February 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Abstract Correcting for sequencing and PCR errors is a major challenge when characterising genetic diversity using high-throughput amplicon sequencing (HTAS). Clustering amplicons by sequence similarity is a robust and frequently used approach, but it reduces sensitivity and makes it more difficult to detect differences between closely related strains. We have developed a multiplexed HTAS method, MAUI-seq, that incorporates unique molecular identifiers (UMIs) to improve correction.
    [Show full text]
  • Mitigating PCR /Amplicon Contamination in a High Risk High
    terial D ac ise b a o s c e y s M Das et al., Mycobact Dis 2018, 8:2 Mycobacterial Diseases DOI: 10.4172/2161-1068.1000261 ISSN: 2161-1068 Research Article Open Access Mitigating PCR /Amplicon Contamination in a High Risk High Burden Mycobacterial Reference Laboratory in a Resource Limited Setting Prasanta Kumar Das*, Somtirtha B Ganguly and Bodhisatya Mandal Intermediate Reference Laboratory, State TB Demonstration cum training Centre Kolkata-10 West Bengal, India *Corresponding author: Prasanta Kumar Das, Intermediate Reference Laboratory, State TB Demonstration cum training Centre Kolkata-10 West Bengal, India, Tel: 9433355536; E-mail: [email protected] Received date: May 11, 2018; Accepted date: May 25, 2018; Published date: June 01, 2018 Copyright: © 2018 Das PK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Background: Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. PCR contamination/Amplicon Contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these Molecular Biology Laboratories has been very crucial over the years. Though closed system PCRs has substantial reduction in the PCR contamination rates the conventional probe based hybridization methods continues to show occurrence of contamination for various reasons. The Study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high burden setting. Bringing out the most effective interventions in controlling PCR contaminations for future endeavors stood as a priority.
    [Show full text]