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QStart identifies transcription start sites (TSS) based on the aligned fragments of the query genome that can be mapped against the reference genome. TSS can be identified using different criteria, including the sum of all aligned fragments supporting the estimate or the average of aligned fragments supporting the estimate. To take account of the fragment coverage on the reads aligning to the genome, fragments with high alignment score are more likely to be identified as TSS, while fragments with less coverage are less likely to be identified as TSS. Both alignment score and coverage of the aligned fragments are accounted for by the estimation of fragment length and of the rate of false positives. In theory, it could be nugatory if the mismatches were removed following the greedy alignment strategy. However, due to the fact that mismatches are always removed following the alignment strategy used in bowtie2, allowing mismatches will not impair the performance of de novo assembly or TSS identification. However, allowing more than one mismatch will be a problem for the mRNA alignment. The '-R' parameter can be used to control the number of mismatches allowed. When '-R' is set to n0-1, the n-mer approach can be specified. In this case, if a read is mapped to a sequence with at least n-1 mismatches, then it will be discarded; otherwise the read is matched if the alignment score is greater than or equal to the defined threshold. While allowing multiple mismatches for the mRNA reads seems to be theoretically correct, it might affect the performance of TSS identification when the sequencing quality is low. Therefore a warning will be issued if the -R parameter is set to n0-1.
The different method for identifying a TSS has different error status. For the sum of fragments approach, only the position of the TSS is estimated, while for the average of fragments approach, the position of the TSS is estimated together with the reliability of the estimate. Therefore, a TSS is identified by a position and a flag indicating the reliability of the estimate. d2c66b5586