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  • Reliability of short insert sizes

    I have recently sequenced a number of PCR amplicons on an Illumina MiSeq, in a sample that must be a compund heterozygote for 2 SNPs (based on phenotype). The 2 SNPs are very close together, so in theory any read that covers both SNPs should have one of the variants, but never both. However, when I physically count them, only around 80% of reads follow this pattern, while the remaining 20% look like they have both variants, or neither.

    The read length was set to 150bp, but quite a few of the reads (in the 20% group that don't appear as expected) are much shorter than that, as short as 40bp (when BAM files are viewed in IGV). Also, in many of the reads from the 20% group, one or both of the SNPs are within about 5 nucleotides of the end of the read.

    I have since realised that the short reads are due to very short fragment/insert sizes to begin with (so the mate pairs are the same sequence/overlap 100%).

    However, will this have an effect on the reliability of the variant calls?
    If not, how do I explain that only 80% of the reads that span both SNPs (around 200 reads), have genotypes concordant with a compound heterozygote?

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