New Highly-Integrated Tools for Sensitive Next-Generation Sequencing Analysis of Circulating Cell-Free DNA

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Peter Hahn, PhDAssociate Director, R&DNGS Technology DevelopmentQIAGEN GmbH

New highly-integrated tools for sensitive next-generation sequencing analysis of circulating cell-free DNA

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Agenda

Circulating cell-free DNA (cfDNA)• Background• Applications

Highly integrated sample and library preps for cfDNA• QIAseq cfDNA all-in-one Kits for Illumina• QIAseq cfDNA all-in-one T Kits for Ion Torrent

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cfDNA in plasma

N1 N2 N3cfDNA preparation

• cfDNA (also called free-circulating DNA) in plasma is fragmented by plasma nucleases

• DNA complexed with histone molecules is protected against fragmentation, which results in a typical nuclease-digestion size pattern (majority ~170bp, 340bp, 510bp)

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Origin of cfDNA in plasma

• In healthy individuals, the majority of cfDNA molecules in plasma are derived from hematopoietic cells

• About 10% of cfDNA in pregnant women is of fetal origin (mainly from placental cells)

• In cancer patients, cfDNA from tumor cells is released into the plasma and can be detected there

◦ Levels of cfDNA can vary greatly depending on individual health and/or therapy status

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Discovery of cfDNA: historical developments

Modified from: Taglauer, E.S., Wilkins-Haug, L. and Bianchi, L.W. 2014. Review: cell-free fetal DNA in the maternal circulation as an indication of placental health and disease. Placenta 35, S64-68

Scientific Investigation Clinical Integration

1948cfDNA detected

in blood

1999Pancreatic

cancer KRAS mutation

2003-2004Placenta

emerges as major source of

cfDNA2008

Use of MPS for NIPT of fetal aneuploidies

2013Increased

awareness of CPM as

explanation for discordant NIPT

results

1997Identification of male cfDNA in

maternal serum

2002Discovery of

uniquely methylated cfDNA regions enable analysis

independent of fetal gender

2004-2005cfDNA alterations

detected in preeclampsia and

preterm labor 2010ctDNA as

potential liquid biopsy

2012ff identified as

significant influence on

cfDNA analysis

MPS: Massively parallel sequencingNIPT: Non-invasive prenatal testingCPM: Confined placental mosaicismff: fetal fraction

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Circulating cell-free DNA (ccfDNA): a hot research topic

*Pubmed (as of April 19, 2016)

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Analysis of cfDNA from tumor cells as liquid biopsy

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Comparison of blood biopsy and tissue biopsy in cancer

McLarty, J.L. and Yeh, C. 2015. Circulating cell-free DNA: The blood biopsy in cancer management. MOJ Cell. Sci. Rep. 2, 21

Key characteristic Blood biopsy Tissue biopsy

Invasiveness No Yes

Sample availability throughout the disease process Yes No

Sample stability when maintained ex vivo Yes Stable when processed

Utility for longitudinal disease monitoring Yes No

Cost Low High

Processing time ShortLong (involves tissue sectioning, staining and pathologists)

Rejection/failure rate Low High (due to QNS or TNI)

Starting material for multiple testing Sufficient Scarce

Table abbreviations: QNS: Quality not sufficient TNI: Tumor not identified

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cfDNA as liquid biopsy to monitor therapy response and resistance

Heitzer, E., Ulf, P. and Geigl, J.B. 2015. Circulating tumor DNA as a liquid biopsy for cancer.Clinical Chemistry 61, 112-123

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Use of cfDNA for non-invasive prenatal testings (NIPT)

• Classical screening methods, such as seroscreening or ultrasound, have high false-positive rates of 5% and 10-15% respectively

• As a result, 1 in 20 women has to decide whether to undergo invasive tests, such as amniocentesis or chorionic villius sampling

◦Invasive procedures involve a risk of fetal loss at rates of 1 in 300 cases

• NIPT that analyze fetal DNA present as part of free-circulating DNA in maternal plasma are alternatives to other prenatal screening methods to detect monogenic disorders and chromosomal aberrations

Hui, L. and Bianchi, D.W. 2013. Recent advances in the prenatal interrogation of the human fetal genome. Trends in Genetics 29, 84-91

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Cell-free DNA sequencing

Circulating cell-free DNA

Library construction

Whole genome sequencing (WGS)

Target amplification

Target hybrid capture Library construction

Whole exome sequencing (WES)

Targeted sequencing

Targeted sequencing

Data analysis & interpretation

An overview of different cfDNA sequencing workflows

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Challenges in cell-free DNA library construction

•Low amount of input DNA◦ 1-100ng/ml plasma

•Amount of cfDNA in serum/plasma is highly variable ◦Varies from person to person◦May also differ within the same person depending on health status

•Small size of cfDNA (majority ~170bp)

Cell-free DNA posts special challenges for library construction

Integrated sample and library prep protocols that work with both low amounts AND wide ranges of input DNA are required

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QIAseq cfDNA all-in-one Kits for Illumina sequencers

Workflow description

Based on gold standard QIAamp technology

Based on QIAseq Ultra Low Input Library Prep Kits

Market-leading library amplification technology (Optional, dependind on the amount of starting material and intended application)

PCR-free cfDNA library construction from 10ng input

2-step, 1 tubeprotocol(55 min)

cfDNA sample prep from plasma

End polishing

Adaptor ligation

Clean-up/adaptor removal

Library amplification PCR

NGS library

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Comparing alternative sample prep protocols

cfDNA was isolated from the plasma of healthy individual donors and a donor pool using three alternative sample prep protocols

• QIAamp protocol with carrier RNA• QIAamp protocol without carrier RNA • QIAsymphony protocol (without carrier RNA)

Isolated cfDNA (1 ng) was subjected to library preparation (four replicates) and sequenced after library QC

• Comparable library yields between different samples and protocols• Very low sample-to-sample variation

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• Similar distribution of mappable reads on each chromosome • The ratio of reads per X and Y chromosome reflects the sex of the donor (pool)

Comparing alternative sample prep protocols

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Robust performance over a broad range of input volumes

Evaluation of different plasma input volumes/cfDNA input in library prep

9.5ng cfDNA from 5ml plasma 5.7ng cfDNA from 3ml plasma

3.8ng cfDNA from 2ml plasma 1.9ng cfDNA from 1ml plasma

Library yield: 139nM in 25µl Library yield: 127nM in 25µl

Library yield: 83nM in 25µl Library yield: 59nM in 25µl

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Comparing amplified vs. non-amplified cfDNA libraries

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Library concentration from 10ng cfDNA is sufficiently high for sequencing without amplification

8 PC

R cy

cles

• Libraries with a concentration of 2nM are fine to freshly dilute samples for whole genome sequencing

• To acquire higher amounts of DNA libraries, PCR enrichment with a limited number of cycles is required

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Similar read distribution per chromosome

Normalized Unique Reads

Comparing amplified vs. non-amplified cfDNA libraries

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Efficient cfDNA library construction using QIAseq all-in-one Kit

• Eight cfDNA samples (DNA input ranging from 20 – 37ng in a volume of 43µl) were used for library prep

• The QIAseq cfDNA all-in-one Kit and a kit from supplier N were used in parallel

• Calculated the conversion rate for each sample

Experimental design

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Hybrid capture for exome sequencing

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Highly uniform coverage

QIAseq cfDNA all-in-one prep kit for target sequencing

• Four cfDNA standards (Horizon) were analyzed in an eight-sample Nextseq run with ~50 million reads/sample (2x 150bp paired-end sequencing)

• Highly comparable mean coverage irrespective of the initial amount of cfDNA used

◦99.7% of all bases had coverage of ≥ 15x◦99% of all bases had coverage of ≥ 40x

• Analysis of mutation levels is ongoing◦(Eight verified mutations on chromosomes 1, 3, 7 and 12)

Starting amount of cfDNA

10ng 10ng 2ng 2ng

≥ 40x coverage 0.99 0.99 0.99 0.99

Mean coverage 194.78 209.31 228.24 199.81

% bases above 15x coverage 99.7 99.7 99.7 99.7

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Use Seraseq Aneuploidy Reference Materials as artificial samples

Reference samples from Seracare can serve as full process controls

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Evaluating QIAseq cfDNA workflow with aneuploidy model systems

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Experimental procedure

Data analysis: T18 used as reference for T21 and vice-versa

Seraseq T18 (12%) aneuploidy reference sample

QIAseq sample preparation (500µl; ~10ng DNA)

Library construction (from 3ng DNA)

Low-coverage whole genome sequencing

Seraseq T21 (12%) aneuploidy reference sample

QIAseq sample preparation (500µl; ~10ng DNA)

Library construction (from 3ng DNA)

Low-coverage whole genome sequencing

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Library qualification and quantification shows high purity and yield for both samples

T18; 12%

T21; 12%

New QIAseq cfDNA all-in-one library prep kits deliver high quality libraries

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Sequencing data metrics

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• Seracare T18 and T21 reference material

• Clearly detectable and highly specific chromosomal aberrations in both samples with relatively low number of reads (4 – 6 million reads/sample)

Evaluating cfDNA workflow using full process aneuploidy samples

Overrepresented chr18 in sample T18 (12%)Overrepresented chr21 in sample T21 (12%)

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cfDNA sample prep from plasma

End repair/adapter ligation/nick repair (upto 56.5 µl cfDNA input)

Clean-up/adaptor removal

Library amplification PCR

NGS library

QIAseq cfDNA all-in-one Kits for Ion Torrent

1-step protocol (35 min)

First one-step shot-gun library prep in market: QIAGEN IP

Coming soon!

Workflow description

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Sample to Insight with cell-free DNA sequencing

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New Highly-Integrated Tools for Sensitive Next-Generation Sequencing Analysis of Circulating Cell-Free DNA