Analysis of Targeted Bisulfite Sequencing Sample Clauses

Analysis of Targeted Bisulfite Sequencing. ‌ The raw sequencing files have low quality reads near the end of the read and adaptor sequences still attached, which should be removed so they do not interfere with the next steps. Fastq sequencing reads were quality trimmed using Trim Galore (Version 0.4.4, available at xxxx://xxx.xxxxxxxxxxxxxx.xxxxxxxx.xx.xx/projects/trim_galore/) and FastQC (Version 0.11.5, available at xxxxx://xxx.xxxxxxxxxxxxxx.xxxxxxxx.xx.xx/projects/fastqc/) (Figure 6 and 7) prior to being mapped to the human genome (GRCh37) using Bismark (Xxxxxxx and Xxxxxxx, 2011) and Bowtie 2 (Xxxxxxxx and Xxxxxxxx, 2012) (Figure 8). Methylation was called and compiled into percent methylation using Bismark. The annotated coverage files were then visualized with the BeeSwarm package in R (R Core Team, 2017). Figure 6: Quality control of raw sequencing files. Reads with low quality scores (<20) can pose threats to future steps including false positives or failure to align reads. The low quality reads that are trimmed are highlighted in the red box. Notice the significant improvement of quality after trimming (vast majority >20). Figure 7: Removing adaptor sequences. Adaptors are artificial sequences that will interfere with read alignment and must be trimmed as well. Here, one can observe that after trimming, all adaptor traces have been removed, as highlighted in the red box (adaptor content ~0%). Figure 8: Read alignment and methylation calling. Reads are in silico converted and aligned to bisulfite converted genomes. When the different alignments are compared, one can determine the context and methylation status therein. In the current study, methylation status in CpG context is selected.