Next-generation sequencing (NGS) coverage describes the average number of reads that align to, or "cover," known reference bases. The sequencing coverage level often determines whether variant discovery can be made with a certain degree of confidence at particular base positions.
At higher levels of coverage, each base is covered by a greater number of aligned sequence reads, so base calls can be made with a higher degree of confidence.
30X coverage is considered the standard industrial quality, a number of studies demonstrates that it is the best compromise between cost and quality. Indeed, increasing coverage after 30X creates a smaller benefit compared to cost increase.
At Dante Labs we guarantee a number of reads that are sufficient for a 30X coverage of a human genome (90Gbases) but in most of the cases we oversequence samples and the average of data given to customer is more than 98Gbases.
Moreover, our sequencing is always associated with a guaranteed quality with ≥ 75% of bases higher than Q30 at 2 x 150 bp, and also in this case we do better than what we promise with an actual Q30 at 2 x 150 bp > than 90%.
Saliva is a good source of DNA for WGS, but an incorrect sampling can however affect the quality of our sequencing in an uncontrolled way.
Saliva is a very good source of human DNA for sequencing, the study by Wall et al. (2014) found “no difference in the sequencing quality or error rate of blood and saliva samples”, but they did not perform a systematic comparison of the coverage, microbial contamination.
In more than 90% of our customers auto-sampling of saliva is performed correctly and bacteria reads represent less than 5% and are not affecting the WGS coverage.
However, in some cases we observed a very low mapping on human DNA and extremely high mapping on non-human DNA. This affects the quality of sequencing by adding an extremely high DNA contamination from non-human sources.
In these cases, Dante Labs does not feel responsible for the low coverage of the sequencing, customers will be contacted to address the possible causes of the low coverage and decide the actions to take.
For your convenience following a list of good practices for ensuring a good Human DNA quality in Saliva samples.
Note: since bacteria reads do not map to the human reference genome and remain unmapped during read alignment, they are not included in downstream analyses and therefore are unlikely to have a major effect on variant calling, VCF and report generation.