Miseq platform

Targeted next-generation sequencing was performed using the MiSeq platform (Illumina, Inc., San Diego, CA, USA) with OncoPanel_AMCv3 (OP_AMCv3, Celemics Inc., Seoul, Korea) to capture the exons of MLH1, MSH2, MSH6, and PMS2. Genomic DNA (200 ng) from normal small intestine mucosa was fragmented to 250 bp by sonication (Covaris Inc., Woburn, MA, USA), followed by size selection using Agencourt AMPure XP beads. A DNA library was prepared by ligation of 50 ng of purified DNA with a TruSeq adaptor using the SureSelect XT Reagent Kit (Agilent Technologies, Santa Clara, CA, USA). Each library was made with sample-specific barcodes 6 bp in size and quantified using PicoGreen, and eight libraries were pooled to a total of 700 ng for hybrid capture using an OP_AMCv3 RNA bait. The concentration of the enriched target was measured by quantitative PCR (Kapa Biosystems, Inc., Woburn, MA, USA), and samples were loaded onto the MiSeq platform for paired-end sequencing. The data were analyzed with a laboratory-developed pipeline for variant calling. Variants with more than 20× depth were considered as true candidates. The found variants from targeted panel sequencing were assessed by the LOVD (LOVD v.2.0 Build 36) and InSiGHT (LOVD v.3.0 Build 17) database and ClinVar archive to identify novel germline mutations.

Total RNA was extracted from 200 μL plasma using the AgencourtRNAdvance blood kit (Beckman Coulter, CA, USA) and then reverse-transcribed using Superscript III (Invitrogen, CA, USA). The preparation of libraries and the sequencing were performed as in a previous study [19 (link)]. Briefly, double-stranded DNA was generated with an NEB Second Strand Synthesis kit (New England BioLabs, Ipswich, MA, USA). After that, the KAPA Library Prep kit (KAPA Biosystems, Boston, MA, USA) was used to prepare libraries and pooled at equimolar concentrations for sequencing on the Illumina MiSeq platform (v3 chemistry). Bioinformatic processing was performed as before [19 (link)]. First, the raw reads were trimmed using trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ accessed on 3 July 2022). Next, the clean reads were enriched in silico by removing the human and rRNA reads (Ribopicker, http://ribopicker.sourceforge.net/, accessed on 3 July 2022) and then matched against whole-genome HCV reference sequences using Tanoti (http://bioinformatics.cvr.ac.uk/tanoti.php, accessed on 3 July 2022). For GT-3b, because there are only 14 full-length sequences in NCBI, these reads were iteratively assembled using the SPAdes de novo assembler [20 (link)]. Finally, the same files were generated using Tanoti, and a majority consensus sequence was generated for sites with >5 reads.

The genomes of the three E. albertii strains CB9786, NIAH_Bird_3, and EC06-170 were sequenced using the Roche 454 GS FLX Titanium platform, 400–500 bp shotgun fragments and 8 kb-span paired end libraries. The sequence reads were assembled with GS Assembler ver. 2.3, and gaps were filled by sequencing fosmid clones and PCR products that spanned the gaps using a capillary sequencer (ABI3730). The three strains were resequenced with the Illumina MiSeq platform to correct sequencing errors made by the Roche 454. The protein-coding sequences (CDSs) and functional annotations were predicted using the Microbial Genome Annotation Pipeline (MiGAP; http://www.migap.org, last accessed November 12, 2015). Manual curation was performed using the in silico Molecular Cloning Genomics Edition software (IMC-GE; In Silico Biology, Inc.).