Shallow WGS workflow

Genomic DNA was extracted from the FFPE samples. 50 µl of DNA samples were mechanically sheared using Covaris ME220 Focused-ultrasonicator. Libraries were prepared from 100 ng of DNA using Agilent SureSelect XT HS and XT Low Input Library Preparation kit (Agilent, Santa Clara, CA, USA, ref: G9703A) according to the manufacturer’s instructions. This process included ligation, PCR amplification, and purification on AMpure XP beads (Beckman Coulter, Indianapolis, IN, USA, ref: A63882). DNA concentration was measured using either Thermo Fisher Scientific Qubit® dsDNA HS Assay Kit (ref: {"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}}Q32854) or Qubit® dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, ref: {"type":"entrez-protein","attrs":{"text":"Q32853","term_id":"75280860","term_text":"Q32853"}}Q32853). Library quality and quantity were assessed using the Agilent TapeStation and D1000 ScreenTape. A pre-capture library pool with concentrations of 4 nM or 1.8 nM was generated for NextSeq 550 S or NovaSeq 6000 Sequencing systems (Illumina Inc, San Diego, CA, USA), respectively.

In Poitiers Hospital, DNA from FFPE samples were extracted on a Maxwell® 16-IVD using FFPE Plus LEV DNA Purification Kit (Promega, Madison, WI, USA, ref: AS1135). 200 ng of FFPE DNA were used for generating pre-capture libraries on a Magnis NGS Prep System using the SureSelect XT HS Low Input kit (ref: G9731D). 100 µl of molecular biology grade water were added to the QC strip wells (blue strip) and quantification was performed with on a Qubit® 3 fluorometer using the 1x dsDNA HS Assay Kit (ref: {"type":"entrez-protein","attrs":{"text":"Q33230","term_id":"75319368","term_text":"Q33230"}}Q33230). The pre-capture library pool was sequenced at 1 nM on a NextSeq 550 platform (Illumina). After sequencing, FASTQ files were generated and analyzed by shallowHRDv2.