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Sample to Insight
Dr. Bhagyashree S. BirlaNGS Field Application Scientistbhagyashree.birla@qiagen.com
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
3Eric Lader 5/3/2018 Human ID
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
4Eric Lader 5/3/2018 Human ID
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
5Eric Lader 5/3/2018 Human ID
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
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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
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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.
8Eric Lader 5/3/2018 Human ID
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
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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
10Eric Lader 5/3/2018 Human ID
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)
Conventional targeted DNA sequencing
EGFR exon 21
11Eric Lader 5/3/2018 Human ID
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
Conventional targeted DNA sequencing
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
* *****
*
12Eric Lader 5/3/2018 Human ID
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
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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
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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
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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
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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
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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
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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.
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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
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Sample to Insight
Thank You!
23Eric Lader 5/3/2018 Human ID
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