TITLE: Low-Pass WGS Sequencing Library Preparation
AUTHORS: Christopher T Boniface

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Simple protocol for generating WGS libraries using KAPA HyperPrep library preparation reagents.</div></div>

[STEPS]
?. [DNA extraction ]
Extract 10-50ng of tumor DNA from FFPE needle-core biopsy or resection using QIAamp DNA FFPE Tissue Kit
?. [DNA extraction ]
Check sonicated DNA size distribution using Agilent Bioanalyzer 2100 system or similar electrophoresis
?. [DNA extraction ]
Quantify extracted DNA and then sonicate 10-50ng of DNA to fragment to ~150bp fragments.
?. [Library Preparation]
Using 10-50 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)
?. [Library Preparation]
During step 4 incubations, anneal TSHTDM adaptor oligos to an appropriate stock concentration (100uM) by combining, in equal parts, i5 (sense) and i7 (antisense) adaptor oligonucleotides being mindful of the index sequences used. Anneal the single-stranded oligos by heating the mixture to 95C for 5 min and letting cool to RT for 30-60 minutes.
?. [Library Preparation]
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.
?. [Library Preparation]
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 20 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 20 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.
?. [Library Preparation]
Ligate 15 minutes at 20C in a thermocycler (lid heat "off").
?. [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.
?. [Library Amplification]
Amplify library using KAPA HiFi Hotstart temperature protocol for 8-12 cycles (60C annealing temperature).
?. [Library Amplification]
Bead purify using 50ul bead solution (1X vol.) washing and eluting as above (step 8).
?. [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).
?. [Library Amplification]
Amplified libraries are now ready for dilution and sequencing using an Illumina NGS platform. 40-80M PE reads is sufficient for 0.5-1X mean WGS coverage for analysis.