Fig 1.
Linked target capture workflow.
(a) Custom adapters (i) are ligated to template DNA and the resulting product is amplified with universal primers. (b) Target regions are selectively amplified using custom probe-dependent primers (PDPs) (ii) which contain a recognition sequence (dark grey) with a 3’ blocker (black diamond) and are linked to an oligo containing a universal priming sequence for the first target capture PCR reaction (tcPCR1). (c) A second set of PDPs (iii), which contain Illumina adapters (red and black) between the probe and linked universal primer, are then added and a second target capture PCR reaction (tcPCR2) is completed prior to (d), clean up and QC and (e) loading on a sequencer. The inset shows detail of forward and reverse PDPs.
Table 1.
LTC 35-gene sequencing performance data for two replicates of sample type and DNA mass.
On-target fraction was defined as the fraction of total bases that aligned to the target regions. Mean target coverage was defined as the mean de-duplicated coverage over all target regions, and uniformity was defined as the fraction of on-target bases that were covered within two fold of the mean target coverage (i.e. between 0.5x and 2x of the mean). Median insert length was measured over all de-duplicated on-target inserts.
Fig 2.
Representative insert size distributions for each sample type used in this study.
Table 2.
The expected allele percentage was measured and specified by the manufacturer using digital PCR or next generation sequencing. Expected allele percentages are given for stock samples (Sample 1), samples diluted to 1/10 of the stock concentration (Sample 2), and wild-type samples (Sample 3).
Fig 3.
Expected vs. measured SNV, indel and fusion fractions.
The dotted line represents a 1:1 ratio of expected vs. measured variants. Undetected and zero variant fraction samples were reported at 0.0% for display purposes.