Nebnext ultra 2 end repair kit

DNA obtained from the ancestral strains and evolved lines that showed PFGE variation were end-repaired (NEBnext ultra II end repair kit, New England Biolabs, MA, USA) and cleaned up with 1× AmPure beads (BeckmannCoulter, USA). Native barcode ligation (BC02-05) was performed with NEB blunt/TA ligase (New England Biolabs, MA, USA) and cleaned with 0.5× AmPure beads. Qubit quantified barcode ligated DNA samples were pooled at equi-molar concentration to attain 1 µg pooled sample. Adapter ligation (BAM) was performed for 15 min using NEBNext Quick Ligation Module (New England Biolabs, MA, USA). Library mix was cleaned up using 0.4X AmPure beads (Beckmann Coulter, USA) and library was eluted in 15 µl of elution buffer and used for sequencing. Sequencing was performed on MinION Mk1b (Oxford Nanopore Technologies, Oxford, GBR) using SpotON flow cell (FLO-MIN107) in a 48 h sequencing protocol on MinKNOW 1.7.7. Base calling was performed using Albacore v.1.2.6. Reads were processed by albacore and poretools (Loman and Quinlan 2014) (link) for converting fast5 files into fasta format.

Detection of HIV-1 ISs was performed using ligation-mediated PCR and high-throughput sequencing, as previously described (Satou et al., 2017 (link); Katsuya et al., 2021 (link)) but with minor modifications. Briefly, cellular genomic DNA was sheared by sonication using the Picoruptor device to obtain fragments with an average size of 300–400 bp. DNA ends were repaired using the NEBNext Ultra II End Repair Kit (New England Biolabs) and a DNA linker (Satou et al., 2017 (link)) was added. The junction between the 3′LTR of HIV-1 and host genomic DNA was amplified using a primer targeting the 3′LTR and a primer targeting the linker (Satou et al., 2017 (link)). PCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen) according to manufacturer’s instructions. This was followed by Ampure XP bead purification (Beckman Coulter). Purified PCR amplicons were quantified using Agilent 2200 TapeStation and quantitative PCR (GenNext NGS library quantification kit; Toyobo). LM-PCR libraries were sequenced using the Illumina MiSeq as paired-end reads, and the resulting FASTQ files were analyzed as previously described (Satou et al., 2017 (link)). A circos plot showing virus ISs in the Jurkat/NL4-3 model and different cell lines was constructed using the OmicCircos tool available as a package in R software (Hu et al., 2014 ).

For PCR based Nanopore library preparation, PCR amplicons using Khc-73 specific primers with barcoding adapter sequences were used. Each sample was barcoded by PCR using Nanopore PCR barcoding kit (EXP-PBC001). Barcoded samples were pooled at equimolar concentration, and end-prepped using NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair Kit. The nanopore adapter was ligated using Nanopore ligation sequencing kit (SQK-LSK109). Alternatively, samples were prepared without barcoding and sequenced separately. In this case, PCR amplicons at the equimolar concentration were end-prepped and the nanopore adapter was directly ligated. MinION Mk1B device and FLO-FLG001 flow cells were used for sequencing of the libraries.