RIPTIDE enabled Next Generation Genotyping...CONFIDENTIAL RIPTIDE Size Selection (Included in Kit) SPRI bead based size selection flexible for multiple insert size ranges a) Pre-size

RIPTIDE™ enabled

Next Generation Genotyping

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Acknowledgements

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Next Generation Genotyping Workflow (Same for all RIPTIDE Applications)

De-multiplexing

Sample Extraction (RNA/DNA) Library Preparation (RNA/DNA) Sequencing

Result: Variant Call File

for each genome

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RIPTIDE Workflow

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RIPTIDE Workflow: 960 Individually Barcoded Samples

A)

B)

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RIPTIDE Size Selection (Included in Kit)

SPRI bead based size selection flexible for multiple insert size ranges

a) Pre-size selected library produces an

equimolar amount of product from 200bp-2kb+.

b) Example of SPRI bead size selected material with

500bp mean insert length. Options for larger/smaller

inserts included in protocol.

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RIPTIDE Data Analysis: Read Structure

Sample Barcode

Random Sequence

Template Bases

Template Bases

8 nt 12 nt 130 nt 142 nt

Random Sequence

8 nt

P5 Adapter

P7 Adapter

Bases identifying the sample

Plate Barcode (Index)

6 nt

Bases identifying the plate

Read 1 (150 nt) Read 2 (150 nt)

Derived from primer A Insert Derived from primer B

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RIPTIDE: One Technology – Many Applications

Whole Genome Sequencing Microbiome Genotyping by Sequencing

Targeted Sequencing

Sample# of

Barcodes

Mean Coverage

(unique)# hets called

% hetsphased

N50 Haplotype

block

NA128781 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

Phasing / Haplotyping RNA Sequencing

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RIPTIDE Generates Uniform Coverage and is Reproducible

Heat map of wheat genomes:

Chinese Spring (Reads/Mbp)

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Riptide Enabled Next Generation Genotyping (NGG): Simple Description

Homozygous (inbred) parental lines

• Shallow sequencing of progeny determines

recombination ”boundaries”

• Known (parental) genotypes are “assigned”

Deep Sequencing of parental lines

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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)

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What about a more

complicated scenario?

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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?

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Human Data Generation

Samples obtained

from Corriel institute

4ul of DNA input into

Riptide (no sample

QC performed)

Multiplex libraries

sent to macrogen

for sequencing

Demux using fgbio…

…then Gencove for

VCF generation for

38M bi-allelic variants

RTGtools VCF eval

used for comparison

ILMN GSA array

variants in NIST high

confidence regions

used as "truth" set

*Gencove now calls > 80M variants for human

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960 Human Genomes Across a Full NovaSeq S4 Flow Cell

Number of samples per flow cell depends on goals of study.

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384 Human Genomes: (96 Per Lane on NovaSeq S4 Flow Cell)

99.7%98.2% 98.9% 98.7%

99.0% 99.6%

92.5%

87.5%

90.0%

99.9%

50.0%

55.0%

60.0%

65.0%

70.0%

75.0%

80.0%

85.0%

90.0%

95.0%

100.0%

SNV Precision SNV Sensitivity SNV Accuracy MAF 5%Precision

SV Precision SV Sensitivity SV Accuracy ReplicatePrecision

Summary Stats (mean) for JPT and YRI samples (n=288) on autosome variant calls

Wellderly (96, right), YRI(96) & JPT(96X2)

Wellderly: NGG vs Illumina 40X WGS Gold Standard

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0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1

Rea

d C

ou

nt

Accuracy: f-measure

0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1

Re

ad C

ou

nt

Sensitivity: TP / (TP + FN)

0.0E+00

2.0E+07

4.0E+07

6.0E+07

8.0E+07

1.0E+08

1.2E+08

1.4E+08

0.9 0.91 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1

Re

ad C

ou

nt

Precision: TP / (TP+FP)

Precision, Sensitivity, and Accuracy for 288 Individual Samples

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Precision (Concordance) by MAF: GSA

98.0%

98.5%

99.0%

99.5%

100.0%

0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 100%

Pre

cisi

on

Minor Allele Frequency

Precision: TP / (TP + FP) by Minor Allele Frequency

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Comparison of Human Genotyping Products

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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

https://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-zhttps://doi.org/10.1038/s41576-018-0016-z

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Reference: University of Liege

“Reagent costs to sequence a mammalian

genome at 1-fold depth are now

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RIPTIDE + Gencove Promotion: Purchase a Kit, Get Your Analysis for Free!

Reference: https://docs.gencove.com/main/#data-analysis-configurations

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Back to technology

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RIPTIDE Tunability and Customization

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RIPTIDE Kit Includes Hi/Low GC Primers for Tunability

• Easily tunable to GC content

• Species representation is maintained

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RIPTIDE targeted spike ins: in development

TTR Gene: 17 targeted primer annealing sites spaced 300-600bp across 9.564kb

IGV browser images

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NGS library (rRNA sequences in purple)

Total cellular RNA sample

NG

S lib

rary

pre

p

CRISPR-mediated Depletion Protocol: (A JumpCode Technology)

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NGS library (rRNA sequences in purple)

CRISPR Cas9 digestion of library

(or multiplexed libraries)

Size selection to remove short fragments

PCR amplfication

Total cellular RNA sample

NG

S lib

rary

pre

p

Post

-lib

rary

rib

od

eple

tio

n


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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)

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0.31%

4.25%

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

Predepletion Post Depletion

% reads coding

15.5

7.3

0

2

4

6

8

10

12

14

16

Predepletion Post Depletion

Genome size (Gbp)

JumpCode’s CRISPR repeat depletion applied to wheat

Reduction of genome size

(78% goal)

53%

Enrichment increase of

CDS coverage Theoretical

max = 8.49%

14x

7.3

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Conclusions

High quality data

Simple, cost effective

Tunable to the data you want

Just the beginning…

RIPTIDE enabled Next Generation Genotyping...CONFIDENTIAL RIPTIDE Size Selection (Included in Kit) SPRI bead based size selection flexible for multiple insert size ranges a) Pre-size

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