Output from Identify Mispriming Events
Four outputs are produced from the Identify Mispriming Events tool:
- Mispriming events: An annotation track that can be used for Trim Primers of Mapped Reads. The track has a row for each mispriming event.
- Primers: An annotation track of primers annotated with different mispriming statistics.
- Misprimed reads track: A read mapping of reads representing mispriming events.
- Report: A report that summarizes the mispriming events identified by the tool.
Mispriming events
The mispriming events track includes the following annotations:
- Primer sequence: The sequence of the primer.
- Primer length: The length of the primer.
- Misprimed length: The length of the mispriming site where the primer sequence aligns.
- Intended target chromosome: The chromosome that the primer was designed for.
- Intended target region: The region that the primer was designed for.
- Similarity %: Similarity percentage between the primer sequence and the sequence of the mispriming site.
- 3' mismatches: The number of nucleotide mismatches before the first match in the 3' primer end.
- Primer part mismatch type: Yes if the mispriming event potentially causes false positives in the primer part of the mapped read, otherwise No.
- Non-primer part mismatch type: Yes if the mispriming event potentially causes false positives in the non-primer part of the mapped read, otherwise No. Only evaluated if a target region track is provided.
Primers
The primer track includes the following annotations:
- Length: The length of the primer.
- Best hit length: The length of the BLAST hit with the highest similarity percentage.
- BALST hits: Number of filtered BLAST hits for this primer.
- Mispriming events: Number of mispriming events for this primer.
- Mispriming events > 80%: Number of mispriming events with a similarity percentage of at least 80% for this primer.
- Mispriming events > 90%: Number of mispriming events with a similarity percentage of at least 90% for this primer.
- Mispriming events with non-primer part mismatches: Number of mispriming events, originating from this primer, that potentially cause mismatches in the non-primer part of the mapped read.
- Mispriming events with primer part mismatches: Number of mispriming events, originating from this primer, that potentially causes mismatches in the primer part of the mapped read. Only evaluated if a target region track is provided.
- Max mispriming similarity %: Maximum similarity percentage among the identified mispriming events for this primer.
- Unique primer: Yes if the primer is unique in the reference genome, No if the primer has a 100% similarity match to another genomic region.
Misprimed reads track
Each mispriming event is represented by two reads in the read mapping: A read with the sequence of the primer aligned to the mispriming site, and a read with the sequence of the mispriming site aligned to the primer design region. Primers with multiple mispriming events will have a read for each mispriming event aligned at the primer design region.
For mispriming events that potentially cause false positives in the non-primer part of the read, two additional reads are included in the read mapping: A read with the downstream sequence of the primer aligned to the downstream of the mispriming site, and a read with the downstream sequence of the mispriming site aligned to the downstream of the primer design region. The mismatches in these reads show the potential false positive variants that can arise from mispriming.
Report
The mispriming event report includes the following information:
- Summary: A summary table showing the number of input primers and input target regions, as well as how many primers that have mispriming events and the types of potential false positives.
- Primers with potential mispriming: The section provides information about the primers for which one or more mispriming events have been found. The number of BLAST hits and the number of mispriming events for each primer are shown as distribution plots, as well as the maximum mispriming similarity percentage for these primers.
- Mispriming events: Different statistics about the BLAST hits and mispriming events identified by the tool.