RIPTIDE ™ enabled Next Generation Genotyping
Acknowledgements CONFIDENTIAL
Next Generation Genotyping Workflow (Same for all RIPTIDE Applications) Sample Extraction (RNA/DNA) Library Preparation (RNA/DNA) Sequencing De-multiplexing Result: Variant Call File for each genome CONFIDENTIAL
RIPTIDE Workflow CONFIDENTIAL
RIPTIDE Workflow: 960 Individually Barcoded Samples A) B) CONFIDENTIAL
RIPTIDE Size Selection (Included in Kit) SPRI bead based size selection flexible for multiple insert size ranges a) Pre-size selected library produces an b) Example of SPRI bead size selected material with equimolar amount of product from 200bp-2kb+. 500bp mean insert length. Options for larger/smaller inserts included in protocol. CONFIDENTIAL
RIPTIDE Data Analysis: Read Structure Read 1 (150 nt) Read 2 (150 nt) Plate Sample Random Template Template Random Barcode P7 P5 Barcode Sequence Bases Bases Sequence (Index) Adapter Adapter 8 nt 12 nt 130 nt 142 nt 8 nt 6 nt Bases identifying the sample Bases identifying the plate Derived from primer A Insert Derived from primer B CONFIDENTIAL
RIPTIDE: One Technology – Many Applications Whole Genome Sequencing Microbiome Genotyping by Sequencing Targeted Sequencing RNA Sequencing Phasing / Haplotyping Mean N50 # of % hets Sample Coverage # hets called Haplotype Barcodes phased (unique) block NA12878 1 1544 153x 2,612,866 99.9963% 16,791,622 Jurkat 6144 21x 2,229,468 99.2557% 2,261,063 NA04510 6144 34x 2,302,366 99.6859% 3,732,649 NA05289 6144 28x 2,165,774 99.5555% 1,149,861 NA11410 6144 30x 2,653,959 99.6975% 4,753,710 NA11629 6144 29x 2,278,703 99.6456% 1,582,026 NA13707 6144 11x 1,646,406 96.7802% 430,602 NA14622 6144 27x 2,318,691 99.8724% 7,255,334 CONFIDENTIAL
RIPTIDE Generates Uniform Coverage and is Reproducible Heat map of wheat genomes: Chinese Spring (Reads/Mbp) CONFIDENTIAL
Riptide Enabled Next Generation Genotyping (NGG): Simple Description Deep Sequencing of parental lines • Shallow sequencing of progeny determines recombination ”boundaries” • Known (parental) genotypes are “assigned” Homozygous (inbred) parental lines CONFIDENTIAL
Empirical Evidence Shows High Concordance to Arrays: Maize Cross: B73 and LH82 lines 0.01x coverage shows > 99% concordance to AFFX 600K 2.4Gbp genome @ 0.01x coverage (.024G)=< $6 per sample* (*Novaseq S4 flow cell, 2x 150, 100K samples per flow cell) CONFIDENTIAL
What about a more complicated scenario? CONFIDENTIAL
NGG for Accurate Human Genotyping Requires a Bit More Evidence (Reads) How much sequencing is needed to accurately determine the correct haplotype and assign (impute) all the genotypes in a given genomic region? CONFIDENTIAL
Human Data Generation 4ul of DNA input into Multiplex libraries Samples obtained Riptide (no sample sent to macrogen from Corriel institute QC performed) for sequencing ILMN GSA array …then Gencove for RTGtools VCF eval variants in NIST high Demux using fgbio … VCF generation for used for comparison confidence regions 38M bi-allelic variants used as "truth" set *Gencove now calls > 80M variants for human CONFIDENTIAL
960 Human Genomes Across a Full NovaSeq S4 Flow Cell Number of samples per flow cell depends on goals of study. CONFIDENTIAL
384 Human Genomes: (96 Per Lane on NovaSeq S4 Flow Cell) Wellderly (96, right), YRI(96) & JPT(96X2) Wellderly: NGG vs Illumina 40X WGS Gold Standard Summary Stats (mean) for JPT and YRI samples (n=288) on autosome variant calls 100.0% 99.9% 99.7% 99.6% 99.0% 98.9% 98.7% 98.2% 95.0% 92.5% 90.0% 90.0% 87.5% 85.0% 80.0% 75.0% 70.0% 65.0% 60.0% 55.0% 50.0% SNV Precision SNV Sensitivity SNV Accuracy MAF <1% MAF 1-5% MAF >5% SV Precision SV Sensitivity SV Accuracy Replicate Precision Precision Precision Precision CONFIDENTIAL
Precision, Sensitivity, and Accuracy for 288 Individual Samples Precision: TP / (TP+FP) 1.4E+08 1.2E+08 1.0E+08 Sensitivity: TP / (TP + FN) 8.0E+07 1.40E+08 Read Count 6.0E+07 1.20E+08 4.0E+07 1.00E+08 Accuracy: f-measure 1.40E+08 2.0E+07 8.00E+07 Read Count 1.20E+08 0.0E+00 6.00E+07 0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 1.00E+08 4.00E+07 8.00E+07 Read Count 2.00E+07 6.00E+07 0.00E+00 0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 4.00E+07 2.00E+07 0.00E+00 0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1 CONFIDENTIAL
Precision (Concordance) by MAF: GSA Precision: TP / (TP + FP) by Minor Allele Frequency 100.0% 99.5% Precision 99.0% 98.5% 98.0% 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 100% Minor Allele Frequency CONFIDENTIAL
Comparison of Human Genotyping Products CONFIDENTIAL
Potential for Single Phase GWAS and Causal Variant Discovery • Merge FAST Q files from individual low pass samples (case/control) • Treat cases and controls as high coverage individual samples • Perform genotype calling • Call novel variants • Identify causal candidates Reference: https://doi.org/10.1038/s41576-018-0016-z CONFIDENTIAL
Reference: University of Liege SNP-based quantitative deconvolution of biological mixtures: application to the detection of cows with subclinical mastitis by whole genome sequencing of tank milk − Wouter Coppieters, Latifa Karim, Michel Georges “Reagent costs to sequence a mammalian genome at 1-fold depth are now <20 € thus making this a cost-effective proposition. As a matter of fact, the method is being deployed in the field in several countries.” CONFIDENTIAL
RIPTIDE + Gencove Promotion: Purchase a Kit, Get Your Analysis for Free! Reference: https://docs.gencove.com/main/#data-analysis-configurations CONFIDENTIAL
Back to technology CONFIDENTIAL
RIPTIDE Tunability and Customization CONFIDENTIAL
RIPTIDE Kit Includes Hi/Low GC Primers for Tunability • Easily tunable to GC content • Species representation is maintained CONFIDENTIAL
RIPTIDE targeted spike ins: in development IGV browser images TTR Gene: 17 targeted primer annealing sites spaced 300-600bp across 9.564kb CONFIDENTIAL
CRISPR-mediated Depletion Protocol: (A JumpCode Technology) NGS library prep Total cellular RNA sample NGS library (rRNA sequences in purple) CONFIDENTIAL
CRISPR-mediated Depletion Protocol: (A JumpCode Technology) NGS library prep Total cellular RNA sample NGS library (rRNA sequences in purple) CRISPR Cas9 digestion of library Post-library ribodepletion (or multiplexed libraries) Size selection to remove short fragments PCR amplfication CONFIDENTIAL
CRISPR-mediated Depletion Protocol: (A JumpCode Technology) Cas9/gRNA RNP formation (10 mins at room temp) Cas9/gRNA digestion of library (1 hr at 37 ° C) Size Selection (0.6X Ampure Beads) PCR Size Selection (0.6X Ampure Beads) CONFIDENTIAL
JumpCode’s CRISPR repeat depletion applied to wheat Genome size (Gbp) 16 53% 15.5 14 12 10 8 7.3 7.3 Reduction of 6 4 genome size 2 (78% goal) 0 Predepletion Post Depletion % reads coding 10% 9% 8% 14x 7% 6% 5% Enrichment 4.25% 4% increase of 3% 2% CDS coverage 1% 0.31% Theoretical 0% Predepletion Post Depletion max = 8.49% CONFIDENTIAL
Conclusions High quality data Simple, cost effective Tunable to the data you want Just the beginning… CONFIDENTIAL
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