Direction and position filters

Many sequencing protocols are prone to various types of amplification induced biases and errors. The 'Read direction' and 'Read position' filters are aimed at providing means for weeding out variants that are likely to originate from such biases.

Figure 25.39 shows an example of a variant that is removed by the 'Read direction' filter. To see the direction of the reads, you must adjust the viewer settings in the 'Reads track' side panel to 'Disconnect paired reads'. Note that variant calling was done ignoring non-specific matches and broken pair reads, so only the 16 intact forward paired reads (the green reads) are considered. In this example there was no intact reverse reads.

Image variantRemovedByReadDirectionFilterV2
Figure 25.39: Example of a variant that is removed by the 'Read direction' filter.

Figure 25.40 shows an example of a variant that is removed by the 'Read position' filter, but not by the 'Read direction' filter. This variant is only seen in a set of reads having a similar start position, while reads that start in a different location do not contain this variant (e.g., none of the reads that start after position 186,641,600 carry the variant). This could indicate the incorporation of an incorrect base during the library preparation process rather than a true biological variant. The purpose of the 'Read position' filter is to reduce the presence of these types of variants. As with all noise filters, the more stringent the setting, the more likely you are to remove false positives and enrich your result for true positive variant calls but comes with the risk of filtering out true positives as well.

Understanding the type of false positive this filter is intended to remove will help you to determine what makes sense for your data set. For example, if your sequencing data did not include a PCR step or hybrid capture step, you may wish to use more lax settings for this filter (or not use it at all).

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Figure 25.40: A variant that is filtered out by the Read position filter but not by the Read direction filter.