dr. bhagyashree s. birla ngs field application scientist ... · 5/3/2018 · count and analyze...

Sample to Insight

Dr. Bhagyashree S. BirlaNGS Field Application [email protected]

Sample to Insight

DNA ApplicationsHuman IDLiquid biopsyBiomarker discoveryInherited and somatic SNPMicrosatellite instabilityTumor mutation burdenMethylomics (gene regulation)Single cell sequencing

RNA ApplicationsGene expressionPathway analysisBiomarker discoveryAllele specific expressionFusion genes in cancerTumor heterogeneityImmune repertoireSingle cell sequencing

NGS spans a broad range of applications

2Eric Lader 5/3/2018 Human ID

Sample to Insight

QIAGEN NGS Portfolio

QIAseq

DNA

WGS

Methylome Single cell

WES Metagenome Targeted sequencing

SNP in/del TMB MSS

Methyl

RNA

WTSmiRNA/small

RNA sequencing

Targeted sequencing

mRNA/lncRNA Single cell Gene fusionsT cell

receptor


Sample to Insight

The problem with patient samples

• Low purity • Cancer cells may only be a minor fraction of total sample

• Heterogeneity• Multiple sub-clones of cancer may be present in one tumor sample

• Rare targets• 1000X coverage is required to get >90% sensitivity to detect ~5% mutation frequency

Whole genome / exome sequencing is expensive and will not yield sufficient coverage


Sample to Insight

What is targeted sequencing?Sequencing a region or subset of the genome or transcriptome

Why targeted sequencing?Not all regions of the genome or transcripts are relevant to a specific study

• Exome Sequencing: sequencing most of the coding regions of the genome (exome). The protein-coding region constitutes less than 2% of the entire genome

• Focused panel/hot spot sequencing: focused on the genes or regions of interest e.g. Clinical relevance – tumor supressor genes, inherited mutations

What are the advantages of targeted sequencing?More coverage per sample, more sensitive detection

• 1 gene copy ~ 3 pg, 3000 copies in 10 ng• Heterogeneous sample 1% tumor cell = 30 copies in 10ng• Every base not covered equally in typical NGS experiment• More samples per run, lower cost per sample• Compatible with less than ideal sample quality – biofluids, FFPE

Targeted sequencing


Sample to Insight

QIAGEN Targeted NGS Portfolio

QIAseq

DNA

WGS

Methylome Single cell

WES Metagenomic Targeted sequencing

QIAseq DNA Methyl

RNA

WTSmiRNA/small

RNA sequencing

Targeted sequencing

QIAseq RNA QIAseq UPX QIAseqRNAScan

T cellreceptor


Sample to Insight

Attribute/parameter

Information level

Cost persample

Coverage achieved

DNA input

No. of samples multiplexed

Whole genome sequencing

3 x 109 bps

$5000

30x

1 µg

1

Whole exome sequencing

5 x 107 bps

$2000

100x

100–200 ng

2

Targeted DNA sequencing

6 x 104 bps

$200

1000x

10 ng

96

Benefits of targeted DNA sequencing

More relevant data

More cost effective

Detect low-frequency mutations

Lower DNA requirements

Higher multiplexing capabilities

Benefits of targeted sequencing

Targeted DNA sequencing delivers accurate information required for precision medicine

Targeted sequencing reduces cost per sample and required DNA input


Sample to Insight

Typical challenges in targeted DNA sequencing

Inability to detect low-frequency mutations

Inefficient enrichment and sequencing of

GC-rich regions

PCR and sequencing errors• Limits sensitivity and accuracy of calling low-frequency variants

o Doesn’t let you confidently call variants down to 1% variant allele frequency (VAF)

Suboptimal uniformity of

enrichment and sequencing

Suboptimal, GC-rich region-incompatible PCR chemistry• Limits comprehensiveness of panel coverage

o Doesn’t let you efficiently sequence clinically-relevant genes such as CEBPA or CCND1 – or clinically-relevant regions such as TERT promoter

Conventional PCR protocols and two-primer amplicon design• Increases variability in coverage across targeted genomic regions

o Causes you to over-sequence to accommodate the under-sequencedo Doesn’t let you call variants in low-depth regions

Mainly due to intrinsic limitations of PCR amplification approaches.


Sample to Insight

Boosting NGS sensitivity with error correction

UMI: Unique molecular indexA tag (barcode) to identify unique DNA molecules

nnnnnnnnnnnn(12 nucleotides long)

Incorporate this random barcode (signature) into the original DNA or RNA molecules before amplification to preserve their uniqueness


Sample to Insight

PCR and sequencing errors (artifacts) limit variant calling accuracy

A mutation is seen in 1 out of 100 reads that map to EGFR exon Cannot accurately tell whether the mutation is:

1. A PCR or sequencing error (artifact)/false positives, or

2. A true low-frequency mutation of 1%

Conventional targeted DNA sequencing

EGFR exon 21

Variant calling based on non-unique reads does not reflect the mutational status of original DNA molecules

*

Boosting NGS sensitivity with error correction


Sample to Insight

Implementing molecular barcoding in NGS

Count and analyze single original molecules (not total reads) = digital sequencing

Five unique DNA molecules Quintuplets of the same DNA molecule (PCR duplicates)

UMIDigital sequencing with UMIs

UMIs before any amplification

Five reads or library fragments that look exactly the same. Cannot tell whether they represent:

1. Five unique DNA molecules, or

2. Five reads of the same DNA molecule (PCR duplicates)


EGFR exon 21


UMI

Sample to Insight

Achieve accurate variant calling with molecular barcodes

Count and analyze single original molecules (not reads) = digital sequencing

Digital sequencing with UMIs

UMIs before any amplification

A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is:

1. A PCR or sequencing error (artifact) / false positives, or

2. A true low-frequency mutations


EGFR exon 21

False variant is present in some fragments carrying the same UMI

True variant is present in all fragments carrying the same UMI

UMI

* *****

*


Sample to Insight

QIAseq

DNA

WGS

Whole Methylome

WES Metagenomics Targeted sequencing

DNA Methyl

RNA

WTSmiRNA/small

RNA sequencing

Targeted sequencing

mRNA/lncRNA Gene fusions Immune

UMIs in the QIAGEN NGS Portfolio

WGS, WES, WTS, Metagenomics sequencing usually too shallow to benefit from UMIsUMIs need to be read more than once for error correction to be effective

The use of UMI increases sensitivity in targeted DNAseqand quantification accuracy in targeted RNAseq


Sample to Insight

SPE technology: DNA variant analysis

Enzymatic fragmentationSPE:

More tolerant of fragmentation than PCR

Random fragmentation yields unique molecules

More tolerant of large primer pools than PCR

Easier to optimize

Unmatched uniformity


Sample to Insight

Specifications of QIAseq targeted DNA panels

DNA input As little as 10 ng DNA

Primer multiplexing level Up to 20,000 primers per single reaction

Enrichment technology Single primer extension (SPE) with UMI

Amplicon size Average 150 bp (tunable enzymatic fragmentation)

Sample multiplexing 384 (Illumina), 96 (Ion Torrent)

Total workflow DNA-> Library 8 hours

Variant allele or fusion frequency called 0.5% across entire panel

Somatic and germline SNP, in/del, CNV, MSI, TMB, mitochondrial genome


Sample to Insight

QIAseq DNA panel performance

High uniformity is critical for low level mutation calling

Panel Panel size (bases) Uniformity (0.2x mean base %)

Pharmacogenomics Panel 3,313 99.34

Actionable Solid Tumor Panel 15,160 99.85

BRCA1 And BRCA2 Panel 16,405 100

Mitochondria Panel 16,570 99.08

BRCA1 And BRCA2 Plus Panel 25,590 99.92

Colorectal Cancer Panel 215,328 99.79

Lung Cancer Panel 318,059 99.91

Breast Cancer Panel 370,942 99.84

Myeloid Neoplasms Panel 436,672 99.71

Comprehensive Cancer Panel 836,670 99.76

Inherited Diseases Panel 838,627 99.21

TMB Panel 1,500,000 99.8


Sample to Insight

Content for a wide range of applications

QIAseq targeted DNA panels; Raed N. Samara, PhD 17

Cancer

Breast cancerColorectal cancer

Myeloid NeoplasmsLung cancer

BRCA1 & BRCA2BRCA1 & BRCA2 plus

Actionable solid tumor

Comprehensive cancer

Pharmacogenomics

Mitochondria

Inherited disease

Extendable

Custom

Your own:GenesExons

Hotspots/SNPsIntronic regions

Easily increase panel content with SPE primers

Sample to Insight

RNA NGS logisticsDynamic range: 1 to 100,000 copies of any one RNA in a cell

With a 105 range, read budget has to be high enough to capture targets of interest

Targeted RNA NGS allows the rational design of a panel to maximize value and throughput

Further complexitiesEven within a ‘pure’ cell population there can be significant cell-to- cell variability in gene expression.

QIAseq Targeted RNA Sequencing

Bulk analysis of RNA expression represents a steady state average of a complex, dynamic sample

18

RNAseq

Celle/tissue

FFPE

Single Cell

CTC

Biopsy

Biofluids

Sample to Insight

Targeted gene expression: Why NGS?

• HEK293T Cells treated with 90 different small molecule inhibitors.• One day from RNA to sequence ready libraries for 96 samples. • Overnight run on NextSeq

cells

treated cells

RNA

Indexed libraries

Normalized, pooled libraries


Sample to Insight

QIAseq RNAscan: fusion gene detection and classification

A fusion gene is a hybrid gene formed from two previously separate genes. It can occur as a result of: translocation, interstitial deletion, or chromosomal inversion.

20

Sample to Insight

RNAscan workflow: cDNA synthesis, then SPE

Eric Lader 5/3/2018 Human ID

Sample to Insight

Sample isolation

Library construction and target

enrichment

NGS run Data analysis Interpretation

RNA panels

DNA panels

RNAScan

QIAseq UPX

Biomarker development and pathway analysis

Mutations, indels, copy number variants, Human ID

Fusions in disease

Single cell high throughput analysis

QIAseq targeted NGS – a complete Sample to Insight solution


Sample to Insight

Thank You!


dr. bhagyashree s. birla ngs field application scientist ... · 5/3/2018 · count and analyze...

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