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Tim Mercer Genome In A Bottle - Sept 16th

Representing the human genome with synthetic spike-in controls.

DISCLAIMER: The Garvan Institute of Medical Research has filed patentapplications on some techniques described in this study.

Tim Mercer Garvan Institute for Medical Research

Human Genome

Reverse Genome

Human Genome

5’ to 3’Synthetic Genome

3’ to 5’Human Genome Reverse Genome

5’ to 3’ 3’ to 5’

less than 1% Cross-Alignment

(low-complexity sequences)

HUMAN (FWD)simulated

(101nt, paired)

SYNTHETIC (REV) simulated

(101nt, paired)

NA12878(Illumina platinum

genomes,101nt, paired)

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MapQ score

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MapQ score

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HUMAN GENOME (5’ to 3’) SYNTHETIC GENOME (3’ to 5’)LIBRARY:

Read

Alignments

Split-Reads

Discordant

Alignments

Duplication Duplication

Human Genome

5’ to 3’Synthetic Genome

3’ to 5’

NGS reads from human genome and the mirror genome have the same alignment properties (direction agnostic).

Human Genome (5’ to 3’) Reverse Genome (3’ to 5’)

SPLICED GENES

FUSION GENES

IMMUNE RECEPTORS

GENETIC VARIATION

PRIMER SITES

STRUCTURAL VARIATION

REPEAT DNA

ONE COPYHALF COPY HALF COPY

RNA DNA

Size-selection

Purification

In Vitro Transcription Digestion

Size-selection

Purification

Sequin Manufacture RNA sequins (left) by in vitro transcription and purified. DNA sequins (right) by restriction digestion, and purified.

Expected Abundance

Ob

serv

ed A

bun

da

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Expected Abundance

Ob

serv

ed A

bun

da

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Mix A

Mix B

Mixture A Mixture B Fold-difference

Variable Sequins(Measure differences between samples)

Constant Sequins(Normalise between samples)

Mixtures Individual (RNA or DNA) sequins are combined to emulate quantitive features (eg. gene expression, splicing, allele frequency) and establish internal reference ladders.

Expected Abundance

Observ

ed

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Observ

ed

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un

da

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Expected AbundanceMix A

Mix B

Mixture A Mixture B Fold-difference

Variable Sequins(Measure differences between samples)

Mix A Mix B Fold-Change

Mixture Accuracy We can measure the variation between five replicates due to: 1) Independent sources (due to mixture prep.) ~0.027sd 2) Dependent sources (sequence specific etc.) ~0.285sd

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1.0

1.5

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Average normalized sequin abundance

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Mix 2Mix 3Mix 4Mix 5

Systematic variationIndependentvariation (pipetting)

Independent variation (pipetting)

Mix 1

Sequins are added to a RNA/DNA sampleat a fractional concentrations (typically 2-3%).

The combined sample is then sequenced, with a proportional fraction of the reads derived from sequins in the final library.

To distinguish reads in the library that derive from sequins, we align the library to a combined index comprising both the human genome (hg38) and also the mirror genome.

Human Genome

Synthetic Genome (Reversed)

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BRAF V600E

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Re-reversing partitioned alignments visualised synthetic genome features in the same direction as the human genome.

GENETIC VARIANTS

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Sequence Read

Coverage

Alignments

Identified Variants

Homozygous Variation

Heterozygous Variation InDels

Sequin A

Sequin B

in silico ChromosomeVariant A

Variant B

Homozygous Variation

Heterozygous Variation

Sequin B

Sequin A

Manufacture sequins

Combine with genome DNA

for sequencing and analysis

1kb length240 SNPs/Indels sampled from dbSNP(Deveson et al., Nature Methods 2016)

v2 (available shortly)

1.8kb length99 SNVs/Indels sampled from NA12878 high confidence (v2)99 SNVs/Indels in difficult regions(high & low GC, mono/di/tri nucleotide repeats)heterozygous / homozygous as per annotation

v1

Library Preparation

Reference GenomeSynthetic Genome

SampleSequins

Sequencing

Alignment

Analysis Results

Example Workflow

Sequins added (~2%) to NA12878 genome DNA sample prior to library preparation.

Undergo sequencing (125nt paired-end Illumina to ~40x coverage).

Calibrated Coverage Subsample sequin alignments to calibrate precisely calibrate coverage (left, blue) with the matched regions in the accompanying human genome (right, red).

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Length (Percentile) Length (Percentile)

Edge effects Edge effectsC

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Sequins Human Genome (NA12878)

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Median Per-Base CoverageMedian Per-Base CoverageSe

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(%)

Sens

itivi

ty (%

)

Sequins

NA12878

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Sequin SNVSequin Deletion

NA12878 SNVNA12878 Deletion

Single Nucleotide Variation (SNV) Insertion/Deletion (Indel)

Sequins

NA12878

Germline Variation Synthetic heterozygous variants detected comparably to human variation (using the NA12878 reference annotations) across range of sequence coverage (1-50x).

Somatic Mutations By titrating ‘variant’ sequins relative to ‘reference’ sequins, we can establish the range of somatic mutation frequencies observed in complex tumour sub-populations.

Sequin Frequencies

FOXP 1/FLT3

FLT3/IDH1

CXCL17

TP53

IDH2/RUNX1

Griffith et. al., Cell Systerms 2015

Sensitivity Assess quantitive accuracy of measuring allele frequency with NGS: 1) Limit of Quantification indicates the minimum allele frequency required for accurate

quantification. 2) Correlation and slope describe quantitive accuracy and biases of NGS assay.

Het

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Expected Allele Frequency (log2)

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n Intercept: -0.0619612Slope: 1.08278R2: 0.943421

Precision Detection of false positive variants (from sequencing error, misalignments etc.) in sequins enables an estimate of specificity (precision).

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Sequence Read

Coverage

Alignments

Heterozygous Variation

Sequin A

Sequin B

True PositiveFalse Positive

(Sequencing or alignment error?)

Sequins are a simple and effective method tomeasure diagnostic power of NGS ibrary.

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1:1,0241:2,0481:4,096

AlleleFrequency

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1.00001.00000.99620.99341.00000.99681.00000.98340.93820.64080.54950.16830.1173

AUCvalue

Precision (Cumulative Fraction)

Sensitivity (True-Positive)

Precision (False-Positive)

Cum

ulat

ive

Frac

tion

False-Positive Rate

True

Pos

itive

Rat

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Expected Allele Frequency (Log2)

Test Precision Test Diagnostic

RnaAlign

(Alignment performance)

RnaExpression

(Gene, Isoform and Exon Expression)RnaFoldChange

(Differential Gene Expression)

plotLinear

(Gene Expression)plotLOD

(Fold-change sensititivty)plotROC

(Fold-change sensititivty)

RnaSubsample

(Calibration of Multiple Samples)

RnaAssembly

(Isoform Assembly)plotLogistic

(Isoform Assembly)

Library Preparation(polyA, ribo-depletion etc.)

Next-GenerationSequencing

User’s RNASample

RNA SequinControls

Combined Sample(with 2-3% sequins)

Spike In

.FASTQ

ANAQUIN in C++

ANAQUIN in R

LABORATORY PROTOCOL

Alignment(eg. BWA,BowTie2,Tophat2,STAR)

Gene Assembly(eg. Cufflinks,StringTie)

Normalisation

Gene Expression(eg. Cufflinks,Kallisto,

DESeq2,edgeR)

.BAM,.SAM

.BAM*,.SAM*

.VCF,.TXT

.GTF,.TXT

RNA-SEQ BIOINFORMATICS PIPELINE

Human Genome(hg38)

In SilicoChromosome

x

y

DiagnosticStatistics

Inter-SampleNormalisation

ReferenceLadders

OutputReport

AssessPerformance

ANAQUIN - SEQUIN ANALYSIS TOOLKIT

Anaquin software toolkit for the analysis of sequins that integrates with NGS analytical pipelines, supports standard formats and common bioinformatic tools.

SEQUINS ARE FREE FOR NON-PROFIT RESEARCH, request an aliquot from www.sequin.xyz

Acknowledgments: Ted Wong

Jim Blackburn Ira Deveson

Bindu Kanakamedala Simon Hardwick

Wendy Chen James Ferguson

John Mattick Katrina Frankcombe

Peter Whitfield

Further Reading ‘Representing genetic variation with synthetic DNA standards.’

by Deveson et al., (2016) Nature Methods

‘Spliced synthetic genes as internal controls in RNA sequencing experiments’ by Hardwick et al., (2016) Nature Methods