TITLE: An error-corrected panel-based next-generation sequencing assay for ultra-sensitive somatic mutation detection in ctDNA
AUTHORS: Heidi Fettke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Circulating tumour DNA (ctDNA), fragmented DNA shed into the circulation from tumour cells, holds the potential to become a minimally-invasive biopsy material with which to interrogate the tumour landscape. However, ctDNA can be highly diluted in normal cell-free DNA (cfDNA) (released into the blood primarily by hematopoietic cells), and thus robust liquid biopsy assays with high analytical sensitivity are required for ctDNA to reach its full clinical potential in molecular genotyping.</div><div class = "text-block">This protocol uses optimised off-the-shelf reagents in order to generate indexed and molecularly-tagged cfDNA and germline DNA libraries compatible with Illumina short-read sequencing. This protocol involves the hybridisation-capture of targeted regions of the genome using RNA baits - and has been used previously with a 42-gene panel 447 kbp in size. Also described here is the bioinformatic tools used for data processing and variant identification. </div><div class = "text-block">The combination of deep-sequencing and error-correction based on molecular barcoding allows high sensitivity and specificity to be generated at low variant frequencies. </div></div>

[STEPS]
?. [Blood collection]
Collect whole peripheral blood in either an EDTA tube or a dedicated cfDNA stabilising tube such as a Streck Cell-Free DNA BCT tube depending on the estimated time to processing (within 4 hours for an EDTA tube otherwise use a stabilising tube). 21-23 gauge needle or larger preferred to reduce chances of haemolysis. Invert tube ~5 times, then try to prevent any further agitation during transit if possible.
?. [Blood processing]
Centrifuge blood tubes at  to facilitate the initial separation of plasma from other blood components.
Centrifuge: 1900 34, 10 min, 4 10
?. [Blood processing]
Separate plasma into  eppendorf tubes, being careful not to draw up the buffy coat layer below.At the same time, collect the buffy coat layer (approx  ) below the plasma into a cryovial and store at  until required.
5 ml
1 ml
-80 °C
?. [Blood processing]
Further clarify the plasma via centrifugation at  .Draw up supernatant into a fresh  eppendorf tube, leaving any pellet behind to be discarded.Re-centrifuge the supernatant to ensure the absence of a pellet.Aliquot the plasma into cryovials and store at  required.
Centrifuge: 16000 34, 10 min, 4 10
5 ml
-80 °C
?. [cfDNA extraction]
Thaw plasma on ice.Extract cfDNA from plasma using the QIAamp circulating nucleic acid kit (Qiagen) as per the manufacturer's instructions. Elute into  buffer AVE. Pass the eluate over the column again to maximise yield after a second on-column incubation period of   . Note: Try to avoid extracting cfDNA from more than 6 patient samples at once in order to avoid cross-contamination as much as possible.
50 µl
?. [cfDNA extraction]
Remove contaminating large molecular weight DNA fragments (that can arise due to cellular lysis during the blood draw or transporting process:- Incubate the  eluate with ampureXP paramagnetic beads (Beckman Coulter) (a 0.5:1 v/v ratio) at room temperature for .- separate beads on a magnetic rack for  and draw off the supernatant into a new eppendorf tube. The beads now contain large DNA fragments (over 500 nt) and can be discarded- to the supernatant, add a further  ampure XP beads (1:1 v/v) to the supernatant and incubate for  at room temperature in order to capture the remaining cfDNA fragments- separate beads on a magnetic rack for  and discard the supernatant - keeping the beads on the magnetic rack, wash twice with  80% (v/v) pure ethanol- after discarding the ethanol, allow the beads to dry for between   to  (do not leave for so long that cracks appear)- elute the cfDNA from the beads by mixing well with  pure water and incubating at room temperature for - place tube back on the magnetic rack to separate the beads, drawing up the eluate and storing in a new eppendorf tube
50 µl
25 µl
50 µl
200 µl
13 µl
?. [cfDNA extraction]
Assess size distribution, quality and quantity of resulting cfDNA using automated gel electrophoresis (2200 TapeStation and Expert software, Agilent) and the Qubit dsDNA high-sensitivity assay (Thermo Fisher Scientific). When using Qubit, it is a good idea to always use a control i.e.  of standard #2 Note: cfDNA can be stored at either  or
1 µl
4 °C
-20 °C
?. [gDNA extraction]
Thaw buffy coat at room temperature.Extract germline DNA from buffy coat (containing peripheral blood mononuclear cells) using the QIAamp DNA blood mini kit (Qiagen) and elute into  buffer AE.
200 µl
?. [gDNA extraction]
Shear extracted gDNA to 150-200 bp in length using ultrasonication (Covaris), with the following settings:Duty Factor: 10%Peak Incident Power: 175Cycles per Burst: 200Treatment Time: 360 secondsBath temperature: 4-8ºC
?. [gDNA extraction]
Quantify and qualify fragmented gDNA using automated gel electrophoresis (2200 TapeStation and Expert software, Agilent) and the Qubit dsDNA high-sensitivity assay (Thermo Fisher Scientific).
?. [Library construction]
Use the ThruPLEX Tag-seq kit as per the manufacturer's protocol to generate molecularly-tagged libraries from both cfDNA and gDNA. The ideal input is the kit maximum of  as it will maximise the number of unique DNA fragments and improve chances of low frequency variant identification; however as little as  can also be used.
50 ng
10 ng
?. [Library construction]
Clean-up the library fragments by using a 1:1 (v/v) ratio of AmpureXP beads (Beckman Coulter) and eluting in  pure water. Quantify using the qubit as before, and confirm library size (main peak should have moved from ~170 bp to ~340 bp.
33 µl
?. [Hybridisation capture]
Use the Agilent SureSelect target enrichment system (Integrated Sciences) to capture target regions of interest using a library of biotinylated RNA baits complementary to the regions of interest (Integrated Sciences). In addition to the reagents required, you will also need 4 blocking oligonucleotides (IDT) to allow compatability with the SureSelect libraries:Thruplex p5-UMI-11nt extAATGATACGGCGACCACCGAGATCTACACAGGCGAAGACACTCTTTCCCTACACGACGCTCTTCCGATCTXXXXXXTCAGTAGCTCA/3block/Thruplex p5-UMI-8nt extAATGATACGGCGACCACCGAGATCTACACAGGCGAAGACACTCTTTCCCTACACGACGCTCTTCCGATCTXXXXXXGTAGCTCA/3block/Thruplex p7-UMI-11nt extCAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTXXXXXXTCAGTAGCTCA/3block/Thruplex p7-UMI-8nt extCAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTXXXXXXGTAGCTCA/3block/Note: ‘X’ represents either sample-unique barcode or unique molecular identifier (UMI) sequence regions.
?. [Hybridisation capture]
Follow the manufactuer's protocol for the first capture, incubating the baits with the libraries for  at ; with the following alterations:1. Replace SureSelect Block 3 with  of each of the indexing blockers, and 0 pure water (total volume of blocking mix is )2. Use 1 ul of capture library instead of the  suggested for libraries 3. Perform 4 washes with  of Wash Buffer 2, incubated for  each time4. Re-suspend library in  pure water
65 °C
0.5 µl
0.6 µl
7.6 µl
2 µl
400 µl
14 µl
?. [Hybridisation capture]
Perform PCR enrichment of the capture libraries using all   of the captured library as follows:  AB1  Reagent    Volume for 1 reaction<b style="mso-bidi-font-weight:
  normal">  2  Nuclease-free water    19.5µl  3  5x Herculase Rxn Buffer (clear cap)    10µl  4  Herculase II Fusion DNA Polymerase (red cap)    1µl  5  100mM dNTP Mix (green cap)    0.5µl  6  10µM Illumina P5 primer    2.5µl  7  10µM Illumina P7 primer    2.5µl  8  Total    36µl  Run the following steps on the thermal cycler:  ABCD1  Step    Number of cycles<b style="mso-bidi-font-weight:
  normal">    Temperature<b style="mso-bidi-font-weight:
  normal">    Time  2  Step 1    1    98ᵒC     2 mins  3     Step 2  16  98ᵒC     30 secs  457ᵒC30 secs572ᵒC1 min6  Step 3    1    72ᵒC    10 mins  7  Step 4    1    4ᵒC    Hold
14 µl
AB1  Reagent    Volume for 1 reaction<b style="mso-bidi-font-weight:
  normal">  2  Nuclease-free water    19.5µl  3  5x Herculase Rxn Buffer (clear cap)    10µl  4  Herculase II Fusion DNA Polymerase (red cap)    1µl  5  100mM dNTP Mix (green cap)    0.5µl  6  10µM Illumina P5 primer    2.5µl  7  10µM Illumina P7 primer    2.5µl  8  Total    36µl
ABCD1  Step    Number of cycles<b style="mso-bidi-font-weight:
  normal">    Temperature<b style="mso-bidi-font-weight:
  normal">    Time  2  Step 1    1    98ᵒC     2 mins  3     Step 2  16  98ᵒC     30 secs  457ᵒC30 secs572ᵒC1 min6  Step 3    1    72ᵒC    10 mins  7  Step 4    1    4ᵒC    Hold
?. [Hybridisation capture]
Complete the capture protocol by cleaning up the PCR enriched libraries with AmpureXP beads and elute in 33 ul pure water. Quantitate libraries using the Qubit as above. Libraries can be stored at  for up to one week or at  for longer periods.
4 °C
-20 °C
?. [Hybridisation capture]
Perform DCAP-Seq by repeating the hybridisation capture and PCR enrichment procedures above with the following changes:1. Incubate library with probes for  at  instead of 24 hours2. 5 PCR amplification cycles instead of 163. Resuspend final libraries in   pure water
65 °C
33 µl
?. [Library sequencing and data processing]
Generate between 50 -70 million 100-150 bp paired end reads (for cfDNA), and approximately 1 million reads (for gDNA) on the Illumina HiSeq2500 on rapid mode.
?. [Library sequencing and data processing]
Demultiplex libraries using per sample indices and map to the human reference genome (hg19) using BWA-MEM (v 0.7.12) and converted to BAM files for further analysis using Samtools (v 1.6).
?. [Library sequencing and data processing]
Generate read families and perform error-correction using the molecular barcodes using Connor (v 0.5). Only reads with perfectly matching barcodes and at least 3 replicates should be clustered together as a family to increase the validity of each base call. A consensus frequency of at least 0.66 is required to generate a collapsed read.
?. [Variant identification and filtering]
Identify somatic variants by using VarDict (v 1.5.8) in paired samples mode using standard filter settings, with the exception of minmum variant frequency, which is reduced to 0.001%.
?. [Variant identification and filtering]
Filter resulting VCF files using VarSeq (Golden Helix) using the following settings:Somatic variants1. Status matches 'strong somatic'2. Variant not located in intergenic regions3. Variant must either be loss-of-function or a missense variant with a CADD PHRED score of 20 or greater4. Variant not present in gnomAD Exomes Variant Frequencies v 2.1.1 (BROAD) database at greater than 0.001 frequency, or is missing altogether5. Minimum read depth of 500X6. Minimum variant allele frequency of 0.00257. Minimum of 3 supporting reads8. Variant must have at least 1 supporting reverse read and 1 supporting forward readGermline variants 1. Status matches 'germline'2. Minimum read depth of 8X in the germline sample
?. [Variant identification and filtering]
Assess all remaining variants on Integrative Genomics Viewer (IGV) in order to remove any sequencing artefacts or common variants that may have been missed by gnomAD
?. [CNV identification and filtering]
Generate CNV calls per patient sample using VarSeq CNV Caller ( VS-CNV) using the following settings:1. Set up the reference files by importing BAM files from germline (normal) samples that have undergone sequencing using the same targeted panel and methodology as the tumour samples. At least 30 references are preferred. Pre-deduplicated files may be preferred if post-connor depth is low2. Open VarSeq project and calculate coverage regions using the BED file used for panel design3. Generate CNV calls: as standard settings are not sensitive enough to allow cfDNA analysis, custom parameters need to be set on the CNV calling algorithm:- maximum allowable difference between tumour and reference samples - mean z-score for duplications reduced to 1.2; and mean ratio reduced to 1.3- mean z-score for heterozygous deletions reduced to -1.2; and mean ratio reduced to 0.854. Additional filters used are as follows, but may need to be optimised for different assays:- Duplications require a p-value 0.25 AND a minimum target depth of 50- Deletions required to fulfill one of the following:    Have p-value     Have p-value
?. [Library construction]
If you have >400 ng of library, proceed to the next step. If not, perform 1-2 cycles of PCR enrichment following the protocol in Step 16 below.