Minimum total mapped reads per pool
If the panel contains multiple primer pools, it is important to compute QC metrics per each pool separately. Minimum number of total valid mapped reads per pool is an additional QC metric for RNA Fusion panels with multiple primer pools. Total valid mapped reads per pool is the count of all reads assigned to any target in each pool. If any of the pools have total valid mapped reads less than the specified threshold, that pool does not pass QC.
Note: We recommend that you set a total mapped reads for the fusion assay depending upon the expected number of total reads per barcode which is based on the sequencing platform, chip type and number of barcodes per run.
For example : For a sample sequenced using PGM 318 chip multiplexed using 8 barcodes, we expect more than 100000 reads per sample. Our recommended threshold in this case is 20000 minimum total valid mapped reads.
A 20,000 minimum threshold is recommended to avoid the possibility of missing a real fusion (a false negative). 20,000 mapped reads provide acceptably sensitive fusion detection. At that coverage, fusions calls are reliable. However, a real fusion at low abundance may be missed.
Below 20,000 mapped reads, the assay may lack sensitivity, and we recommend repeating the experiment if possible (if sufficient original sample is available). In addition, the 3'/5' Imbalance number is less reliable for very low mapped reads.
The assay is highly sensitive, and if a gene fusion isoform is detected, it is highly likely to be truly present in the sequencing reads. If a fusion is detected in a sample with a low number of total mapped reads, it is highly likely to be a true positive. However, if a sample has a low number of mapped reads, a real fusion at low abundance in the sample relative to the expression control genes may be missed. The limit of detection is lower with larger numbers of mapped reads.