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RIPTIDE™ enabled
Next Generation Genotyping
CONFIDENTIAL
Acknowledgements
CONFIDENTIAL
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.
CONFIDENTIAL
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
CONFIDENTIAL
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)
CONFIDENTIAL
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
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?
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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?
CONFIDENTIAL
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
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)
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
CONFIDENTIAL
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
CONFIDENTIAL
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
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
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
CONFIDENTIAL
Reference: University of Liege
“Reagent costs to sequence a mammalian
genome at 1-fold depth are now
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
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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
TTR Gene: 17 targeted primer annealing sites spaced 300-600bp across 9.564kb
IGV browser images
CONFIDENTIAL
NGS library (rRNA sequences in purple)
Total cellular RNA sample
NG
S lib
rary
pre
p
CRISPR-mediated Depletion Protocol: (A JumpCode Technology)
CONFIDENTIAL
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
CRISPR-mediated Depletion Protocol: (A JumpCode Technology)
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
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
CONFIDENTIAL
Conclusions
High quality data
Simple, cost effective
Tunable to the data you want
Just the beginning…