Question: Can somatic mutations or low frequency variants be detected?
Answer: Long Ranger has not been optimized to detect low frequency variants, although it is theoretically able to detect some variants down to an allele frequency of ~10%. Long Ranger requires at least two reads to call a variant. The expected number of reads covering a position can be calculated from the starting DNA mass (1.25 ng), average molecule size (50 kb), and sequencing depth (30x):
- 1.25 ng loaded DNA --> 0.5 ng total DNA captured in GEMs.
- 0.5 ng translates to about 150 copies of the human genome (also known as molecule or physical coverage).
- The recommendation is to sequence to 30x coverage (also known as read coverage).
- (30x coverage /150 copies) = 0.20x average coverage per molecule.
- At 10% minor allele frequency, you might expect to have 150 x (10%) = 15 copies of the molecules with the minor allele.
- 15 molecules * (0.2x average coverage/molecule) = 3 reads.
- Long Ranger requires at least two reads to call a variant.
However, keep in mind that the sequencing coverage is not even across the genome. Therefore, Long Ranger will be able to detect SOME variants that are 10% allele fraction, but the sensitivity will not be high.