Such a combinatorial scheme reduces the cost for generating adapters with unique barcodes, while the varying length of barcodes increases library diversity at the 5' and 3' ends.
The index sequences were designed such that the FseI site is not re-generated upon adapter ligation.
First, smRNAs were ligated with a 5′ adapter F: and a 3′ adapter to generate cDNAs.
After sequencing, data were processed using the Ion Torrent platform-specific pipeline software Torrent Suite to generate sequence reads, trim adapter sequences, and remove poor signal-profile reads.
Data outputs obtained by semiconductor sequencing were processed using Torrent Suite version 3.6.2 (Life Technologies, Carlsbad, CA, USA) to generate sequence reads, trim adapter sequences, remove poor signal-profile reads, align to the hg19 human reference genome, analyze coverage, and call variants.
Here, we demonstrate that Hi-Plex applied with hybrid adapters can generate a library suitable for sequencing with both the Ion Torrent and the TruSeq chemistries and that adjusting primer concentrations improves coverage uniformity.
The procedure is based on a 5' adapter generating a local-double-strand structure with the transcript.
Trimming the 6-mer adapter generated 51-mer reads.
After removing 3' fragments with magnetic beads precipitation, Illumina adapter 2 was introduced at 3' ends of the tags to generate tag library with different adapters at both ends.
Prior to aligning, we employed Trimmomatic [ 51] to generate clean reads, removing Illumina adapter sequences.
