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Reference mapping and variant detection

Reference mapping and variant detection. Peter Tsai Bioinformatics Institute, University of Auckland. R eference mapping. The mapping is the process of comparing each read with the reference genome. There are many different software available to perform reference mapping

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Reference mapping and variant detection

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  1. Reference mapping and variant detection Peter Tsai Bioinformatics Institute, University of Auckland

  2. Reference mapping • The mapping is the process of comparing each read with the reference genome. • There are many different software available to perform reference mapping • Multiple placement of reads (multi-hits) • Allow gaps • Don’t allow gaps at all • Limits on number of mis-matches • Assess your mapping results • % of total reads mapped • % of uniquely mapped reads • Coverage statistics, variance in depth

  3. Mapped read depth

  4. Variant detection • Identification of point mutation, short insertion and deletion. • We go thought every column of the alignment and see how many alleles are found and how many are different to the reference genome. Reference: ACGAAACGTAGTGAGGAC-GTA sample: ACCAAACGTAGAGAGGACCGTA SNP SNP indels

  5. Complexity of variant detection • 2nd generation sequencing is NOT single molecule sequencing • Due to the PCR amplification, some DNA fragments will be sequenced more often than others => results in uneven coverage across the genome. • This would provide false support in variant detection, as we are usually more confident in variants that has higher coverage support. • Solution: Mark or remove exact duplicate reads when doing variant detection.

  6. Complexity of variant detection • Cloning process artifacts (e.g. PCR induced mutations). • Error rate associated with the sequence reads. • Error rate associated with the mapping. • Reliability of the reference genome.

  7. Calling a variant • A hard cut-off in percentage of difference to reference base. • 75% as minimum threshold for a variant to be call homozygous variant. • Percentage based cut-off assumes you have sufficient coverage. A (ref): 0% G: 100% A (ref): 7% T: 93%

  8. When to call a variant ? A: 18% C: 0% G: 55% T: 27%

  9. Alignment considerations • Perform local realignment and calculate mapping score to determine which one is better.

  10. What depth do I need ?

  11. Factors to consider • Read length • Longer reads are more likely to be mapped with high confidences • Sequencing depth • Require sufficient depth, ~30x • Base call quality for each supporting bases • Use high quality bases, Q30 • Mapping quality • Local realignment to improve variant calling

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