2.8. Next-generation sequencing of LASSO libraries
Paired-end NGS for inversion PCR products Non-sheared Inversion PCR
products were sent for paired-end NGS as per manufacturer’s protocol (2
× 150 bp NextSeq 550 System Sequencing Illumina). Raw paired end
sequencing outputs (R1 and R2 files) from Illumina NextSeq were
processed and analyzed using a computational pipeline to quantitate
distribution of properly formed (concordant) probes over improperly
formed (discordant) probes depending on how read pairs align
to the probe library reference sequences (N = 3087). Primer sequences
were removed using Trimmomatic-0.36. [35] The
reads were then aligned to the LASSO design sequences using the BLAT
tool. [36] The results were tallied using standard
linux tools.