Dna pcr free library prep kit

Whole‐genome sequencing libraries were prepared with the Illumina DNA PCR‐Free Library Prep Kit according to the manufacturer’s instructions. The prepared libraries were sequenced and the high‐quality reads were aligned to human reference genome hg19 using BWA‐MEM (version 0.7.15).¹³, ¹⁴ Sentieon version 201911.01 (Sentieon, Inc., https://www.sentieon.com/) was applied for variants detection.¹⁵ Subjects’ whole‐genome sequencing BAM and corresponding VCF files were uploaded into LifeOmic’s (Indianapolis, IN, USA) Precision Health Cloud (PHC) for PGx analysis. Aldy version 3.0 was operationalized in the PHC to genotype pharmacogenomic star alleles and diplotypes for CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP3A4, and CYP3A5.¹⁶ In the PHC, the JupyterLab notebook was used to extract genotypes of interest not covered by Aldy from the VCF files. Additional details are provided in Supplementary Methods.

All P. pacificus strains were grown on nematode growth medium (NGM) plates before DNA extraction. We rinsed the plates with M9 buffer and collected worm pellets by slow centrifugation at 1300 rpm for 3 min at 4°C. DNA extraction was performed as described previously (Rödelsperger et al. 2017 (link)). PCR-free libraries were generated with a TruSeq DNA PCR-Free Library Prep Kit following the manufacturer's protocol, and sequencing was performed on Illumina MiSeq. Initial assemblies were constructed with the DISCOVAR de novo assembler (version r52488). We checked for Escherichia coli contamination by BLASTN against in-house and NCBI E. coli genomes and removed contaminated contigs after manual inspection. Finally, scaffolding was performed with SSPACE_Basic_v2.0 (Boetzer et al. 2011 (link)) using four mate-pair libraries of sizes 1.5, 3, 5, and 8 kb (that were generated with a Nextera Mate Pair Sample Preparation Kit). The BUSCO software (version 3.1.0 with the odb9 nematode data set) was run in genome mode to assess the completeness of the genome assemblies and for the comparison with phylogenomic data of 54 other nematode genomes (Rödelsperger 2021 (link)).