Kun:LabNotes/ExonomeSeq/bisFirstE8k

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bisFirstExon[edit]

  • Athurva selected 7174 first exons, and designed probes to capture bisulfite converted DNA.
  • A total of 11673 pairs of capturing arms were designed. Some were missing due to various reasons (low local sequence complexity, repetitive regions, etc.).
  • Since there was no restriction on the number CpG sites per capturing arm in the probe design, iterating all combinations of methylation status leads to over 300k probes.
  • I decided to synthesize probes that target 100% methylated and 100% unmethylated templates for two reasons:
    • CpG methylation has a bimodal distribution. Most sites are either methylated or unmethylated.
    • We can treat the DNA with methyltransferase.
  • This is the Perl Script for probe assembly. A total of 19,956 probes were assembled.
  • For proof-of-concept, I only select a subset of probes to fill up the free space of 4069 sequences in the current order. To select a more interesting subset, I first sorted all probes based on the target coverage, then sorted the top 6413 probes with >=70% target coverage based on GC ratio. The top 4069 probes target exons with GC ratio of >=0.608.