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.