Best practices for variant calling in clinical sequencing

• https://www.ebi.ac.uk/sites/ebi.ac.uk/files/content.ebi.ac.uk/materials/2014/140217_AgriOmics/dan_bolser_snp_calling.pdf

Common artifacts in NGS alignments that gave rise to a false-positive de novo mutation call in a family trio. Each pane is an IGV screenshot of WGS alignments for the proband (top track), mother, (middle track), and father (bottom track). Each sample’s track comprises two parts: a histogram of the read depth and the reads as aligned to the reference sequence. Reads are colored according to the aligned strand (red = forward strand; blue = reverse strand).

a False positive associated with low base quality. Most reads supporting the variant have low base quality indicated by lightly shaded non-reference bases. Four reads in the proband showed the alternate allele with good quality, triggering the variant call.

b False positive due to misalignments near the start or end of reads. Notice that the alternate allele is only observed at the start/end of reads in the proband. In this case, the read depth histogram provides a clue as to the cause of the misalignment. As shown in the next panel, this occurs at the breakpoint of a large paternally inherited deletion.

c The same position as in b, but with soft-clipped bases shown in color. BLAT alignment of such reads reveals that the soft-clipped portion matches the other side of the deletion segment some 5.2 kb downstream.

d False positive associated with strand bias. All but one variant-supporting reads in the proband are on the reverse strand, whereas reference-supporting reads are equally represented on both strands.

e False positives associated with low-complexity sequences. In this case, reads erroneously showing a single-base deletion (horizontal black line) at a T-homopolymer are enriched in the proband. R supporting insertions (purple) are also seen. Note that this position is zoomed out compared to the other panels, a recommended practice to visualize the end of repetitive sequences.

f False positives due to paralogous alignments of reads from regions not well represented in the reference. Alignments for proband include reads with several substitutions relative to the reference sequence within the 41-bp viewing window. This typically occurs when reads from sequences not represented in the reference are mapped to the closest paralog

https://media.springernature.com/full/springer-static/image/art%3A10.1186%2Fs13073-020-00791-w/MediaObjects/13073_2020_791_Fig2_HTML.png?as=webp

https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-020-00791-w/figures/2

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