Kapa ltp library preparation kit

For nuclear target sequencing, a total of 500 ng was fragmented to 400-bp fragments with a Bioruptor^® ultrasonicator (Diagenode, Liège, Belgium). Library preparations were performed following de La Harpe et al. (2019) (link) and using a KAPA LTP library preparation kit (Roche, Basel, Switzerland) for sample cleaning, end-repair, and A-tailing steps, and the protocol of Meyer and Kircher (2010) (link) for adaptor ligation and adaptor fill-in reactions steps. Four μl of the ligated fragment solution were amplified for eight cycles using KAPA HiFi DNA Polymerase (Roche, Basel, Switzerland) and the set of 60 dual index primers described in Loiseau et al. (2019) (link). Libraries were quantified with a Qubit^® Fluorometer v2.2 before pooling in equimolar ratio. Target capture was performed using the custom kit PopcornPalm developed by de La Harpe et al. (2019) (link), and targeting 4,051 palm genes. Target capture was conducted on pools of 50 or 51 samples, following myBait^® Custom Target Capture Kits protocol v3.0 (Arbor Biosciences, Ann Arbor, MI, United States), with 18 h of incubation time at 65°C and 12 cycles of post-capture PCR reactions. The pooled target capture reactions were quantified with Qubit^® Fluorometer v 2.2, before sequencing with an Illumina HiSeq 3000 sequencer (Illumina, San Diego, CA, United States) in paired-end 2 × 150-bp mode.

The custom capture oligo set was designed by the SureDesign platform from Agilent Technologies (SureSelectXT Custom Capture Oligo) against SCN9A, SCN10A, and SCN11A. This set was specifically designed to capture all exons, the proximal promoter sequence, as well as limited intron sequences and untranslated regions near exons. The target-captured sequencing libraries were constructed using the KAPA LTP Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA) combined with a SureSelectXT Reagent Kit (Agilent Technologies), and sequenced on an Illumina Hiseq 2500 sequencer (Illumina, Inc, San Diego, CA) at read length of paired-end 2 × 100 bp. Targeted exome sequencing was performed on an initial cohort of 46 IBD patients that included a mixture of individuals with and without inflammation and abdominal pain. Generated reads were aligned with the GRCh37 human reference genome using the Burrows-Wheeler alignment (20 (link)). Variant detection and analysis were performed using the GATK Best Practice for germline SNP/indel finding workflow (Broad Institute). ANNOVAR software (21 (link)) was used to annotate the variants and identify synonymous, non-synonymous, and deleterious variants for further analysis.

Genomic DNA was extracted from blood samples of participants and sheared on a Covaris S220 (Covaris, Inc., Woburn, MA) to generate 200‐bp fragments. Sheared fragments were end‐repaired, A‐tailed, and adapter‐ligated using the KAPA LTP Library Preparation Kit (Kapa Biosystems, Wilmington, MA) according to the manufacturer's instructions. Dual‐SPRI size selection by Agencourt^® AMPure^® XP beads (Beckman Coulter, Inc., Brea, CA) was used to select approximately 340‐bp adapter‐ligated fragments. Pools of four amplified libraries were captured to the custom SeqCap^® EZ Library (Roche NimbleGen, Inc., Madison, WI) following the manufacturers’ instructions. Libraries were paired‐end sequenced (2 × 100 bp) using the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA).

Sequencing libraries were prepared following the SureSelect version 1.7 target enrichment system (Agilent, Santa Clara, CA, USA) [33 (link)]. Briefly, 50 μL of DNA was sheared acoustically using an LE220 sonicator (Covaris, Brighton, UK). Fragmented DNA was end-repaired, A-tailed, and adaptor-ligated using the KAPA LTP library preparation kit (KAPA Biosystems, Wilmington, DE, USA). The resultant HCMV-enriched libraries were then indexed and sequenced on a MiSeq (Illumina, San Diego, CA, USA) with v3 chemistry to yield 300 × 300 nt paired-end reads datasets.

40 ng of each plasmid DNA was sheared into approximately 350 base pair in length by sonication using a Covaris Sonicator LE220 (Covaris). Fragmented DNA was uniquely index tagged with NEBNext Multiplex Oligos for Illumina (New England Bio-Labs, E7780S and ES7600S). The Kapa LTP Library Preparation Kit (KAPA Biosystems, Roche7961880001) was deployed in this process. Libraries were quantified and quality controlled with Qubit dsDNA HS kit (ThermoFisher) and Agilent 4200 Tapestation System (Agilent). Equimolar amounts of each library were pooled together and sequenced on the Illumina MiSeq platform using MiSeq Reagent Micro Kit v2 (2x 150-cycles). Plasmid sequences were assembled using SPAdes v3.10.1 with multiple k-mer sizes. Minimum depth of 100 reads and Phred quality of 30 were used for consensus calling of the assembled sequences.