ccfdna in the lab: optimizing purification for sequencing

Peggy GulleyUniversity of North Carolina

[email protected]

ccfDNA In the Lab: Optimizing Purification for Sequencing

Disclosures

• No relevant financial relationships with Promega Corporation or any commercial interests in any of the instruments/products discussed in today’s presentation

• Promega has provided reagents and technical support for this project

• Plasma DNA as a tumor marker

• cfDNA sequencing panel methods

• Pre-analytic variables:− collection− processing to separate plasma− storage− extraction− quality metrics https://en.wikipedia.org/wiki/Buffy_coat

Topics– Plasma DNA preparation for Sequencing:

Cell Free DNA Preparation

Pre-analytic Factors• Phlebotomy technique

• Anticoagulant type, volume

• Transport & Storage

• Cell separation

• Interfering substances

• Extraction:

− cfDNA length

− elution buffer/ volume

− automation

• Quantity & quality of cfDNA

DNA fragment length

http://www.northark.edu/academics/areas-of-study/health-and-medical/phlebotomytechnician

https://en.wikipedia.org/wiki/Buffy_coat

http://blog.fisherbioservices.com/avoiding-hemolysis-in-blood-sample-collection-and-processing

Pre-Analytic Interferences

• Collection tube• Needle bore diameter >21 gauge• Blood draw through port vs direct venous• Nucleases• Contaminated reagents or equipment

gross hemolysis

leukocyte lysis

Technical• Post-exercise• Inflammation

Natural

SerumEDTA plasmaPlasma preservative

https://imagebank.hematology.org/image/61350/cll--smudge-cells?type=upload

https://www.labce.com/

• Temperature

• Humidity

• Aliquots

• Storage containers and pipettes– low DNA absorption plastics– carryover prevention– calibrated pipettes

• Freeze/thaw cycles

• Labels, specimen identification

• Automation

https://www.amazon.com/Thermo-Scientific-94052150-Filtered-microliter/dp/B00551Q4V8https://www.amazon.com/Globe-Scientific-3001-Polypropylene-Self-Standing/dp/B008DI6IWK/ref=sr_1_fkmr0_1?s=industrial&ie=UTF8&qid=1523889000&sr=1-1-fkmr0&keywords=dna+storage+tube+low+absorption

Specimen Storage of whole blood, plasma, DNA

Extraction of plasma nucleic acid

Target: cfDNA, RNA, microRNALength Elution buffer/volume

nucleosomalcfDNA monomer

dimer

trimer

Apoptosis

Dinucleosome~340bp

~170bp Mononucleosome

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｛✄✄ ✄

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087838

Cell Free DNA SequencingIn Oncology Research

After the surge of cfDNAfollowing initiation of radiotherapy,cfDNA half-life is 3.8 days

H&E stain

EBER in situ hybridization

After surgical resection,ctDNA half-life is 139 minutes

Vira

l Loa

d, p

lasm

a

To EW eta al, CCR 2003, PMID: 12960110 Lo YM et al, CCR 2000, PMID: 10811107

Epstein-Barr Viral cfDNA Load in Plasma During Treatment for Nasopharyngeal Carcinoma

Cell Free DNA in Plasma Reflects Systemic Cancer Genotypes

Lowes LE et al, Int J Mol Sci 2016, 17: 1505

At least one tumor marker is identifiable in most cancer tissues

The ‘Plasma Mutation Panel’ permits non-invasive detection & quantification of tumor markers

Monitor treatment response & clonal dynamics

TimeSymptoms

Tumor Burdenin Plasma

• Monitor tumor burden (efficacy of therapy); predict recurrence

• Detect selected drug resistance mutations or emerging clones

• Identify druggable variants when biopsy is not feasible or safe

• Distinguish germline from somatic variants

Clinical Indications for “Plasma Mutation Panel”in a patient of known cancer genotype

Sequencing

INFORMATICSCollapse barcoded reads,

Align, Call, Interpret, Report variant levels & viral loads

(Unique molecular identifier added to each template; each strand separately amplified)

Adapted from from ArcherDx

Amplicon Sequencing

Analytic Method for NGS: University of North Carolina ‘Plasma Mutation Panel’

Technology features:1) commercial reagents & software support inter-laboratory

harmonization2) both strands are sequenced to confirm variant calls3) “unique molecular identifiers” & varying amplicon lengths:

a) improve precision for quantifying natural DNA variants, b) assure adequate diversity of input DNA

4) high analytic sensitivity & specificity by virtue of:a) noise reduction algorithms and statistical analyses to

distinguish true positive from noise at each position,b) fragmented DNA is preferentially amplified, limiting

interference from leukocyte DNA

Collapse PCR replicates; eliminate in vitro polymerase errors

Apoptosis or necrosis

Dinucleosome~340bp

~170bp Mononucleosome

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｛✄✄ ✄

Specimen EDTA blood, 10mLDNA extraction Promega Maxwell® RSC Instrument, Maxwell® RSC ccfDNA Plasma

Kit or Maxwell® RSC LV ccfDNA Kit on 1 to 5mL EDTA plasmaLibrary preparation Archer Reveal ctDNA 28 modified to target pathogen genomes

(EBV, HPV)Sequencing Illumina NextSeq with v2 Mid reagentsQuality checks ThermoFisher Qubit, KAPA, Agilent Tape StationControls SeraCare, HorizonInformatics Archer Analysis 5.1 software with QA metrics

System components are marketed For Research Use Only. Not for Use in Diagnostic Procedures.

Impact of storing EDTA blood at ambient temperature

before separating plasma

24 hour Delay in Processing EDTA BloodTo Separate PlasmaYields ~11% more DNA

DNA extraction: Promega Maxwell® RSC Instrument & Maxwell® RSC ccfDNA Plasma Kit

Quantification: Qubit® dsDNA HS Assay


Image from Dennis O’Malley MD


Sequencing read statistics are not consistently impactedby the delay


Sequencing read statistics are notconsistently impactedby 24hr delay

We hypothesize our Library Prep method selects for short DNA fragments, to limit the impact of leukocyte lysis

Delay in Processing EDTA Blood Risks False Negative Mutation Calls on low input DNA

Delay in Processing EDTA Blood Risks False Negative Mutation Calls on low input DNA

What we learned:

• EDTA is a suitable anticoagulant for this NGS assay

• Separate plasma from blood cells on the day of collection

• Aim for ~60ng DNA input to library prep

• Choice of Library Prep method may help overcome the effect of leukocyte lysis ex vivo

Large Volume Plasma Extractions

Comparative effectiveness of 2 different extraction chemistrieson the Promega Maxwell® RSC Instrument

• Maxwell® RSC ccfDNA Plasma Kit (“standard” method) on 1mL of plasma• Maxwell® RSC LV ccfDNA Kit (Large Volume/“LV” method) on 1 to 3mL of plasma

DNA recovery was measured by fluorometry using the Qubit® dsDNA HS Assay

Automated pre-extraction yet to be tested

UNC ‘Plasma Mutation Panel’ is Sensitive and Linear for quantifying 23 engineered cancer gene variants in 5 mock plasma specimens

TP53_p.R273H

ERBB2_p.A775_G776insYVMA

EGFR_p.D770_N771insG

TP53_p.S90fs*33

TP53_p.S90fs*33

TP53_p.R273H

ERBB2_p.A775_G776insYVMA

KIT_p.D816V

NRAS_p.Q61R

PIK3CA_p.E545K

PDGFRA p.D842VPDGFRA p.S566fs*6PIK3CA p.E545KPIK3CA p.H1047RPIK3CA p.N1068fs*4RET p.M918TTP53 p.C242fs*5TP53 p.R175HTP53 p.R248QTP53 p.R273HTP53 p.S90fs*33

23 known variants AKT1 p.E17KBRAF p.V600ECTNNB1 p.T41AEGFR p.D770_N771insGEGFR p.E746_A750delELREAEGFR p.L858REGFR p.T790MERBB2 p.A775_G776insYVMAIDH1 p.R132CKIT p.D816VKRAS p.G12DNRAS p.Q61R

del ins SNV

1/800 = 0.125%

1/100 = 1%

1/50 = 2%

1/400 = 0.25%

1/200 = 0.5%

*Seraseq ctDNA v2 Reference Material (SeraCare) is a series of 2mL mock plasmas containing fragmented DNA engineered to have variants at 0.125, 0.25, 0.5, 1, or 2% variant allele fraction

DNA extraction- Promega Maxwell® RSC Instrument & Maxwell® RSC ccfDNA Plasma Kit on 2mL mock plasmaAnalytics- Archer Reveal ctDNA 28 (modified), Illumina NextSeq v2 Mid reagentsInformatics- Archer Analysis 5.1 software with noise reduction and QA metrics

System components are marketed For Research Use Only. Not for Use in Diagnostic Procedures.

Preanalytic Metadata (for purposes of ‘data sharing’)

Collection and TransportBlood Tube Type: EDTA, Acid Citrate Dextrose (ACD), Streck, Paxgene…Blood volumeTransport Temperature from collection site to processing siteFacility, Collection date/time, Specimen IDSpecimen ID, relevant clinical information, timing (e.g. pre/post treatment)Purpose (clinical or research, human subjects permissions)

Blood processingBlood Fractionation Method, list fractions retained (full protocol)Hours from blood draw to completion of processingInterferences (e.g. hemolysis)

AliquotsVolume of and number of aliquotsAliquot container type, specify matrix or additiveStorage temperature, humidity if pertinent

Extraction and PurificationPurification method (full protocol) Date or time of storage retrievalStorage conditions of purified material (tube type, temperature, humidity)

QuantificationQuantification method (protocol for electrophoresis/fluorometry/PCR ladder) Quantity or quality indicators (specify units of measurement)

Adapted from BloodPAC.org

Analytic “Methods Validation” of Massive Parallel Sequencing Panel

• Devise, refine and lock down test system protocol• Test mock specimens in appropriate matrix• Test patient specimens of known genotype/phenotype

Establish acceptable specimen typesTest all the common, actionable genotypesTest the range of types of variants (SNV, indel, CNV, homopolymer, fusions, human/pathogen) - Study and resolve discrepancies by comparator methods- Identify interferences (e.g. chemical, physical, or informatic, such as

improper nomenclature, maximum detectable indel size)• Devise Quality Control for every step that fails at significant rate

Set limits on QC acceptability • endogenous controls • exogenous controls (false positive & negative calls, linearity)

• Establish process controls • amount and quality of input DNA, library quality, minimum depth of coverage• filters: variant allele fraction, population frequency

• Analytic validity: sensitivity, specificity, linearity, reproducibility; judge acceptability• Clinical validity: indications for testing, use of test results in patient management• Guidance and resources for analytic and clinical interpretation of raw data• Ongoing review after implementation (in-house & published evidence; databases)

Guidance for Validating Molecular TestsMerker JD et al, Circulating Tumor DNA Analysis in Patients With Cancer: ASCO and CAP Joint Review. Arch

Pathol Lab Med. 2018, PMID: 29504834“Molecular Pathology Checklist” and other Accreditation Program checklists, College of American Pathologists,

www.cap.org“Analytical Performance Specifications for Comprehensive Genomic Profiling (M00118 v1) and for Qualitative

Tumor-only Somatic Variant Detection using Circulating Tumor DNA (M00135 v2, with checklist)” www.palmettogba.com

Gargis AS et al, “Assuring the Quality of Next-Generation Sequencing in Clinical Laboratory Practice,” Nat Biotechnol. 2012 30(11):1033-6

Rehm HL et al., “ACMG Clinical Laboratory Standards for Next-Generation Sequencing,” Genet Med. 2013 15(9):733-47

Schrijver I et al., “Methods-Based Proficiency Testing In Molecular Genetic Pathology,” J Molec Diag (2014), 16(3):283-7

Aziz N et al., “College of American Pathologists’ Laboratory Standards for Next-Generation Sequencing Clinical Tests,” Arch Pathol Lab Med (2015), 139:481-93

Richards S et al. “Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.” Genet Med. 2015;17(5):405-24

Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline – 2nd Ed, “CLSI MM09-A2” other CLSI guidelines, Clinical and Laboratory Standards Institute, http://clsi.org

“Next Generation Sequencing (NGS) Guidelines for Somatic Genetic Variant Detection,” and “Guidelines for Validation Submissions of Next Generation Sequencing (NGS) Assays under the NYS Testing Category of Genetic Testing – Molecular,” New York State Department of Health (2015)

Matthijs G. et al, “Guidelines for Diagnostic Next-Generation Sequencing,” Eur J Hum Genet (2016) 24, 2–5Archives of Pathology and Lab Medicine, multiple NGS assay validation articles, 141(6):749-812, June 2017Jennings L, et al, “Recommended practices and principles for validating clinical molecular pathology tests” Arch

Pathol Lab Med. 2009; 133(5):743-755Mattocks CJ, et al. “A standardization framework for the validation and verification of clinical molecular genetics

tests” Eur J Hum Genet. 2010; 18(12):1275-1288“Factors to Consider When Making Benefit-Risk Determinations in Medical Device Premarket Approval and De

Novo Classifications” and other pertinent guidance documents, www.fda.gov

Summary

Specimen preparation is a major component of the total test system for cfDNA sequencing

Careful attention to pre-analytic variables promotes robust assay design and accurate results

Medical literature, regulatory and professional group guidance, and product manufacturers support assays that are analytically sound and clinically useful

ccfdna in the lab: optimizing purification for sequencing

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