TITLE: DIDA-Seq Custom Capture cfDNA Library Preparation
AUTHORS: Christopher T Boniface

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;:UNDERLINE;">D</span><span style = "font-weight:bold;">ual </span><span style = "font-weight:bold;:UNDERLINE;">I</span><span style = "font-weight:bold;">ndex </span><span style = "font-weight:bold;:UNDERLINE;">D</span><span style = "font-weight:bold;">egenerate </span><span style = "font-weight:bold;:UNDERLINE;">A</span><span style = "font-weight:bold;">daptor </span><span style = "font-weight:bold;:UNDERLINE;">Seq</span><span style = "font-weight:bold;">uencing</span><span>, or </span><span style = "font-weight:bold;">DIDA-Seq</span><span>, an ultra-sensitive cell-free DNA sequencing approach for the detection and quantification of circulating tumor DNA in peripheral blood plasma at fractions below the background error-rate of traditional next-generation sequencing (NGS). DIDA-Seq combines unique molecular identifiers (UMIs) and patient-specific custom capture panels to routinely detect ctDNA with mutation allele frequencies at or below 0.02% (or 1 tumor derived DNA fragment in 5k normal fragments at a given genomic locus) with error-rates below 1 base in 10kb-50kb (Butler et al 2019, doi:10.1101/mcs.a003772; Boniface et al 2021, doi:10.3390/diagnostics11010073). This protocol describes the preparation of DIDA-Seq libraries using cell-free DNA, custom DIDA-seq library adaptors and blocking oligonucleotides, and custom hybridization capture panels. It uses reagents developed and manufactured by Roche, LTD and Integrated DNA Technologies, Inc with minimally modified versions of their methods.</span></div></div>

[STEPS]
?. [cfDNA Isolation]
Using 3-10 mL of plasma (as needed, see note below) and NucleoSnap DNA Plasma Kit (Macherey-Nagel), follow manufacturer’s protocol to isolate cfDNA and elute with 50 ul nH2O or TE-low. Store at 4C for several days or freeze for long-term storage.
Yield is highly subject to the physiological state and health of a patient where tumor-burden, treatment type and schedule, as well as physical activity, can greatly influence cfDNA levels. A healthy person might range from 5-30ng cfDNA/mL plasma, and a person with metastatic disease undergoing treatment might have 15-200ng cfDNA/mL plasma. The optimal input amount of cfDNA is >=100ng for the custom capture protocol described here, however a minimum input for library prep can be as low as ~20-30ng (~5-10k haploid genome copies). Lower input quantities require more pre-capture PCR and will therefore result in less than optimal, lower-complexity libraries where assay sensitivity is limited by total number of input genomes. Ligation efficiency is also dependent on input concentration and may suffer with lower input levels (
?. [cfDNA Isolation]
Quantify cfDNA using either Qubit 3.0 (Thermo-Fisher), qPCR using KAPA gDNA Quantification Kit (using 44bp primers only), or by Agilent 2100 BioAnalyzer using HS DNA Kit. Total DNA can range between 15-500+ ng for
?. [End-repair, A-tailing and Ligation of DIDA-Seq Adaptors]
Using 20-100 ng of cfDNA from step 2 and KAPA HyperPrep Kit, prepare and carry out end-repair and A-tailing reaction per manufacturer’s protocol (https://rochesequencingstore.com/wp-content/uploads/2017/10/KAPA-HyperPrep-Kit_KR0961-%E2%80%93-v7.19.pdf)
Note: cfDNA does not require sonication as it is naturally fragmented into ~165bp molecules.
?. [End-repair, A-tailing and Ligation of DIDA-Seq Adaptors]
Prepare dual-indexed, degenerate-tagged, adaptor (DIDA) to an appropriate stock concentration (100uM) by combining, in equal parts, i5 and i7 custom adaptor oligonucleotides such that i5 and i7 oligos contain matching index sequences. Anneal the single-stranded oligos by heating the mixture to 95C for 5 min and letting cool to RT for 30-60 minutes.
?. [End-repair, A-tailing and Ligation of DIDA-Seq Adaptors]
On ice, dilute above annealed adaptors such that 5 uL gives a 200:1, [adaptor]:[cfDNA], molar excess and combine with above end-repair and A-tailing reaction, ligase, and ligation buffer per manufacturer’s protocol. Be careful not to allow above reagents and/or ligation reaction to exceed 4-16C prior to next incubation step.
?. [End-repair, A-tailing and Ligation of DIDA-Seq Adaptors]
Ligate overnight (12-18hrs) at 16C in a thermocycler (lid heat "off").
?. [End-repair, A-tailing and Ligation of DIDA-Seq Adaptors]
Bead-purify ligation reaction: Add 88uL (0.8X vol.) well-mixed, room-temperature SPRI Ampure XP Bead slurry (Beckman-Coulter) and mix thoroughly by pipetting or vortexing. Incubate at room temperature for 10 minutes, separate supernatant from beads using magnet and discard supernatant.  Immediately add 200 uL of 80% EtOH to wash, remove EtOH and repeat wash. After removing second EtOH wash, very briefly spin-down and quickly remove excess EtOH.* Immediately resuspend beads in 25 ul of nH2O.
*Note:  It’s very important not to let beads dry after removal of second EtOH wash. Proceed immediately to spin step, quickly remove remaining EtOH by pipet and resuspend in 25 uL nH2O. It is not critical that every last bit of EtOH is removed - the risk of reduced yield from over-drying greatly outweighs any issues associated with a small amount of EtOH carry-over.
?. [Pre-Capture Library Amplification]
Prepare 50 ul PCR reaction using 25ul KAPA HiFi Hotstart 2x MasterMix and Illumina Library Amplification Primers (10 uM [final]),  20 ul of above post-ligation reaction (from step 7), and 5 uL of nH2O + primers.
?. [Pre-Capture Library Amplification]
Amplify library using KAPA HiFi Hotstart temperature protocol for 8-12 cycles (60C annealing temperature).
?. [Pre-Capture Library Amplification]
Bead purify using 50ul bead solution (1X vol.) washing and eluting as above (step 7).
?. [Pre-Capture Library Amplification]
Quantify using BioAnalyzer 2100 (more accurate quantification can be done with qPCR via KAPA Library Quantification Kit but DNA electrophoresis is necessary to assess molecular size distribution).
?. [4.	Custom Oligo Capture Hybridization (per IDT xGen Lockdown Protocol)]
Follow IDT xGen Lockdown "Tube" Protocol (version 4, pages 15-30, see link) using DIDA-Seq blocking oligos. (manufacturer's protocol: https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/xgen-hybridization-capture-of-dna-libraries.pdf?sfvrsn=ab880a07_16)
Please note the following changes to the manufacturer's protocol:-    Samples may be multiplexed prior to drying down with a speed-vac system (pg 23, step 3).-    When drying samples down, use 1 uL 100 uM adaptor i7 custom blocker and 1 uL 100 uM adaptor i5 custom blocker (2 ul total)-    Hybridization capture baits were purchased through Integrated DNA Technologies, Inc (https://www.idtdna.com/) as xGen Lockdown Probe Pools-    Incubate hybridization reaction overnight  (~16hrs) at 65C.-    Post-library amplification can be done with 12 cycles of PCR using Illumina library amplification primers (step 9, above)
?. [4.	Custom Oligo Capture Hybridization (per IDT xGen Lockdown Protocol)]
Amplified, post-capture DIDA-Seq libraries are now ready for dilution and sequencing using an Illumina NGS platform.
Raw sequencing data (fastq files) alignment and DIDA-Seq error-correction software pipeline is available here:  https://github.com/ohsu-cedar-comp-hub/DIDA-Seq