Fig. 1

Workflow diagram of the PCR-based target enrichment sequencing of HERV-K (HML-2) (PTESHK). The workflow is divided into three phases: target enrichment amplification, library construction and bioinformatic analysis. Genomic DNA is randomly enzymatically digested at 30 °C for 5 min to form DNA fragments. Then, after end-repair and A-tailing, a GAPS adapter, to be used for the amplification, is ligated. Suppression PCR is then performed with specific primers targeting the GAPS adapters and 5′/3′ LTR sequences of HERV-K. Nested PCR with inner primers is then used to increase the amplification specificity. After amplification, PCR products of both the 5′ and 3′ ends are mixed together and cleaned with beads. Another end-repair and A-tailing is performed for the ligation of Illumina sequencing adapters, followed by size-selection. Then the library is ready for sequencing on the HiSeq X Ten platform. After sequencing, all sequencing reads are filtered by Trimmomatic to remove low-quality reads, followed by the discarding of PCR duplicates using Picard. Then paired-end reads are merged based on the overlap before filtering the chimeric reads, including LTR and flanking sequences, with STEAK. After acquisition of the trimmed flanking sequence, the GAPS adapter is cut by Cutadapt, and then those reads that mapped to HERV-K sequences are abandoned after the alignment. Last, the reference loci and non-reference loci are catalogued with help of a BED file. Then analysis is done to assess the method. In addition, a schematic of all the primers specific for HERV-K (HML-2) sequence used for NGS library construction was drawn