Pan-microbial detection using Axiom® Genotyping Solution from Affymetrix T. We b s t e r, 1 P. Ra c k , 1 K. McLoughlin,2 S. Gardner,2 M. Mittmann,1 A. Pirani,1 C. Sheppy,1 C. Jaing,2 L. Bellon,1 T. S l e z a k , 2 M. Shapero1 1 Affymetrix, Inc., Santa Clara, CA 95051; 2 Lawrence Livermore National Laboratories, Livermore, CA 94551

ABSTRACT Axiom® MDA output using CLiMax Background The dynamic interplay between microbial, plant, and animal populations is an ATCC 14579 gDNA active area of study. Agrigenomics-related microbiotas include species from Scan the Axiom® MDA archaeal, bacterial, viral, and eukaryotic origins and can span the spectrum from beneficial to pathogenic roles. With the expansion of sequenced microbial Bacillus cereus pBClin15 Chromosome plasmid genomes, microarrays are well-positioned to capitalize on this information content. Threshold probe intensities 99th Here we present an overview of Axiom® Genotyping Solution and a description of percentile of non-targeting probes the design, development, and testing of an array for microbial detection. Axiom® MDA workflow/CLiMax analysis Two iterations of the algorithm Experimental design Identify possible Make initial targets/Calculate composite model Initial Conditional ® ® Iteration Family Species Target description Axiom Microbial Detection Array (Axiom MDA) contains approximately 1.3 million composite log with score score Bacillus 0 567.1 567.1 Bacillaceae Bacillus cereus ATCC 14579 probe sequences targeted towards more than 12,000 microbial organisms. Probe likelihoods for each no targets cereus Bacillus Bacillus cereus, ATCC 14579 1 176.7 176.7 Bacillaceae sequence parameters including length, Tm, and mismatch tolerance were studied cereus plasmid pBClin15 for optimal discrimination. The array design enables species/strain-level resolution Example of probe-level view from individual samples using a Composite Likelihood Maximization (CLiMax) Add top- Composite Bacillus cereus, plasmid pBClin15, Chan1 algorithm developed at Lawrence Livermore National Laboratory (LLNL). Defined ranking model 12 samples of increasing biological complexity were used to assess array sensitivity target to improved? composite 10 and specificity. Additionally, Axiom MDA was tested for the ability to identify 8 >99th percentile of controls Log2 6 microorganisms from bona fide biological samples, and results were compared to between 95th and 99th percentiles intensity <95th percentile orthogonal approaches. Calculate 4 conditional log 1.0 likelihood for 0.8 Conclusions targets 0.6 Axiom MDA offers a solution for the identification and enumeration of microbial 0.4

entities comprising complex biological samples. The unbiased array content design probability 0.2 Hybridization is sample-type agnostic and thus has utility in the context of both plant and animal No improvement/Hit pre-determined set 0 51015 number of cycles — Report predicted targets health and food safety. Furthermore, flexible sample throughput enabled by 24- Target sequence position (KB) and 96-array formats with manual (24 and 96) or automated (96) workflows allows To the left is a schematic of CLiMax, which yields a model of array data of likely target the processing of hundreds of samples per week with minimal manual intervention. sequences present in a sample. To the right is an example of output from two iterations This feature makes Axiom MDA a cost-effective means for detection and of CLiMax from a sample containing B. cereus (ATCC 14579) identifying both chromo- examination of microbiomes. somal and plasmid DNA present. The probe-level view is displayed for pClin15 plasmid and shows probe responses as a function of position of a target sequence of interest. Axiom® biochemistry and workflow Axiom® MDA limit of detection . Genomic DNA or cDNA derived from RNA genomes is isothermally amplified and Sample Input randomly fragmented into 25 to 125 base pair (bp) fragments, enabled through 1 ng 10 ng 100 ng 100 ng To test the limit of detection of the complete None either manual or automated workflow. Human Human Human Stool Axiom MDA workflow, we used a log dilution 1M . These fragments are purified, re-suspended, and hybridized to customized series of T. maritima (ATCC 43589) genomic 100K Axiom® array plates. (gDNA) adding in 1–1 million genome 10K equivalents either in the absence or presence . Following hybridization, the bound target is washed under stringent conditions to 1K copies addedto

sample of increasing amounts of human gDNA, or 100 remove non-specific background. Each nucleotide is queried via a multi-color 100 ng of gDNA extracted from stool. The heatmap ligation event carried out on the array surface. 10 above demonstrates that Axiom MDA can

® maritima T. . After ligation, the arrays are stained and imaged on GeneTitan Multi-Channel 1 detect 1,000–10,000 copies, depending on (MC) Instrument. T. maritima Correct Species/Strain total sample input mass with correct species Thermotoga Correct Genus and strain resolution, and down to 100 copies No Thermotoga detected at the genus level.

Axiom® MDA enables detection at species-level resolution

Family-level sample description – 16S sequencing Species-level description – Axiom® MDA vs. 16S sequencing

Family Level Calls- Species Level Calls - % Species Level Reads Axiom MDA Axiom MDA - 16S Sequencing Expected Actual Streptococcaceae Streptococcus agalactiae 6.1 Read % Read % Streptococcaceae Streptococcus mutans 4.6 Streptococcaceae 15 12.6 Streptococcaceae Streptococcus pneumoniae 1.3 Clostridiaceae 5 12.3 Clostridiaceae Clostridium diolis 0.01 Moraxellaceae 5 10.7 Axiom MDA Species Level Calls Staphylococcaceae 10 10.2 Moraxellaceae Acinetobacter baumannii NONE Bacteroidaceae 5 9.5 Staphylococcaceae Staphylococcus aureus 4.0 Correct Species and Strain Neisseriaceae 5 7.2 Staphylococcaceae Staphylococcus epidermidis 0.1 Enterobacteriaceae 5 7.1 Bacteroidaceae Bacteroides vulgatus 8.1 Correct Species Neisseriaceae Neisseria meningitidis 6.6 Helicobacteraceae 5 6.5 Correct Genus - Closely Bacillaceae 5 6.2 Enterobacteriaceae Escherichia coli 0.02 related species Lacto bacillaceae 5 5.2 Helicobacteraceae Helicobacter pylori 6.3 Enterococcaceae 5 3.9 Bacillaceae Bacillus cereus NONE Actinomycetaceae 5 2.3 Lactobacillaceae Lactobacillus gasseri 4.3 Rhodobacteraceae 5 2.3 Enterococcaceae Enterococcus faecalis 2.4 Deinococcaceae 5 1.3 Actinomycetaceae Actinomyces odontolyticus 2.0 Propionibacteriaceae 5 1.1 Rhodobacteraceae Rhodobacter sphaeroides 2.2 Pseudomonadaceae 5 0.7 Deinococcaceae Deinococcus radiodurans 1.3 Listeriaceae 5 0.4 Propionibacteriaceae Propionibacterium acnes 1.0 Pseudomonadaceae Pseudomonas aeruginosa 0.5 Listeriaceae Listeria monocytogenes NONE Manual or automated Axiom MDA performance was compared to 16S rDNA sequencing using the same mock target prep community sample (HM-276D, BEI*), which contains a 20 species even mix, normalized by 16S rDNA copy number. On the left is a pie chart showing the expected versus Axiom® ® actual read percentages at the family level from 1.3 million reads from V3-V4 16S GeneTitan ® 24- or 96-array Multi-Channel Instrument (Genewiz MetaVX™) sequencing experiment. On the right are displayed top hits from 20 primary iterations of CLiMax, compared to species-level calls enabled by 16S sequencing. Axiom MDA is able to identify 19 of the 20 samples at the species level, Axiom® MDA content with the outlier at the correct genus (Clostridium diolis vs. Clostridium beijerinckii near- neighbors, considered the same species until 2002). Domain Families Species Target sequences Archaea 31 370 603 Viral cDNA as an input to the Axiom® workflow Bacteria 278 6,904 34,152 Fungi 121 414 659 Reverse Probe responses of RSVA PM probes - Chan 1 The negative strand ssRNA 12 Protozoa 30 143 229 transcribe genome of RSVA (NR-43976, BEI*) was reverse Virus 99 4,637 97,856 10 transcribed with two reverse- Total 559 12,468 133,499 Viral RNA 8 transcriptase enzymes and Probe design informed by used to generate target for 1.3 million 6 Target sequences Axiom MDA. Interquartile targeted probes Log2 intensity range of RSVA perfect match Proof-of-concept studies 4 Viral cDNA Picogram probe responses from the Initial studies on a proof-of-concept array with a subset of species were leveraged to input proof-of-concept array show Reverse Reverse No NTC guide probe design for the full content of Axiom MDA. In addition to the ~1.3 million ® that viral cDNA can be used Axiom Transcriptase A Transcriptase B RT as an entry point into the targeted probes, 60,000 non-targeting with matching physical properties of the workflow targeted set are included for thresholding. Axiom workflow.

For Research Use Only. Not for use in diagnostic procedures. *These reagents were obtained through BEI Resources, NIAID, NIH. (for HM-276D as part of the Human Microbiome Project): © 2016 Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom®, and GeneTitan® are registered trademarks of Affymetrix, Inc. Genomic DNA from Microbial Mock Community B (Even, High Concentration), v5.1H, for Whole Genome Shotgun Sequencing, All other trademarks are the property of their respective owners. Genomic RNA from Human Respiratory Syncytial Virus, A1997/12-35, NR-43976.