This tool uses Bowtie2 software to align single-end reads to a reference genome or sequence set. You need to supply the reads in one or more files. Reads can be in either FASTA or FASTQ format, but all reads files need to be in the same format. You also need to provide a FASTA formatted reference sequence.
After running Bowtie2, Chipster converts the alignment file to BAM format, and sorts and indexes it using the SAMtools package.
Bowtie2 uses heuristics for mapping the reads to the reference genome. This means that the reported mapping is not guaranteed to be the best mapping. Several Bowtie2 parameters affect to the mapping accuracy. However higher accuracy (sensitivity) also requires more computing time. In Chipster you can choose, for both end-to-end and local alignment strategy, the sensitivity level from a set of pre-defined parameter combinations that allow you to tune the balance between the computing time and mapping sensitivity:
| Strategy | Corresponding command line options |
|---|---|
| Very fast | -D 5 -R 1 -N 0 -L 22 -i S,0,2.50 |
| Fast | -D 10 -R 2 -N 0 -L 22 -i S,0,2.50 |
| Sensitive (default) | -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 |
| Very sensitive | -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 |
| Very fast local | --local -D 5 -R 1 -N 0 -L 25 -i S,1,2.00 |
| Fast local | --local -D 10 -R 2 -N 0 -L 22 -i S,1,1.75 |
| Sensitive local | --local -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 |
| Very sensitive local | --local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50 |
In addition to the mapping strategy, the Bowtie2 tool in Chipster allows users to modify many other mapping and output parameters too. Please check the Bowtie2 manual for more detailed description of these parameters.
| Parameter | Corresponding command line option | Description of the parameter |
|---|---|---|
| Quality value format used | --phred33, --phred64 or --ignore-quals | Quality scale used in the fastq-file. |
| How many valid alignments are reported per read | none, -k or --all | By default, Bowtie2 reports only the best aligmnmet of the read (based on the mapping quality\). Optionally, if there are several, equally good alignments, you can choose how many of them should be reported? |
| Put unaligned reads to a separate file | --un | Store unaligned reads to a new fastq file. |
| Match bonus | --ma | Match bonus for a match in local alignment. Default value 2 |
| Maximum penalty for mismatch | --mp | Maximum penalty for mismatch; lower quality = lower penalty. Default value 6 |
| Penalty for non-ACGTs | --np | Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as N. Default: 1. |
| Gap opening penalty for the read | --rdg | Gap opening penalty for the reads. Default value: 5. |
| Gap extension penalty for the reads | --rdg | Gap extension penalty for the reads. Default value: 3. |
| Gap opening penalty for the reference | --rfg | Gap opening penalty for the reference. Default value: 5. |
| Gap extension penalty for the reference | --rfg | Gap extension penalty for the reference. Default value: 3. |
This tool returns the alignment in BAM format and an index file for it (.bai). It also produces a log file, which allows you to see what percentage of the reads align with the selected parameter settings. Optionally also a fastq file is produced for the unaligned reads.