Step 7 : Custom FLT3-ITD calling

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In addition to GATK, Mission Bio uses a custom genotyping method to call internal tandem duplications found in the FLT3 gene. Some panels, e.g., Acute Myeloid Leukemia and Myeloid, have amplicons targeting exon 14 and/or exon 15 in the FLT3 gene. We look for insertions in the FLT3 amplicons and map to the locus.

For each cell, we scan for soft-clips and insertions. All insertions and clippings are considered as possible insertions.

If the total number of reads is greater than a cutoff (10) and the number and the ratio of non-REF reads are greater than a cutoff (4 and 0.1 respectively), the cell is considered to have a non-REF allele.

If the ratio of non-REF (alternate allele) reads to total reads is greater than a cutoff (0.9), a homozygous event is called. Otherwise, it is considered a heterozygous event.

If the cell has enough total reads but not enough ALT reads, it is considered a homozygous reference. Otherwise, it is reported as “no call.”

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