Mapping short reads

Regular mappers

These mapping tools are useful for reads of DNA origin that should map to a continuous stretch of genomic DNA. Some of these tools can tolerate short indels but they're not designed for reads that span a splice junction

One may choose between bowtie version 1 (faster but ignores indels) and bowtie version 2 (slower but performs gapped alignment (i.e., indels)). For a feature comparision, see ​How is Bowtie 2 different from Bowtie 1?

​bowtie version 1

Sample command:

bsub bowtie  -k 1 -n 2 -l 70 --best --sam --solexa1.3-quals /nfs/genomes/mouse_gp_jul_07_no_random/bowtie/mm9 s_7.txt s7_mm9.k1.n2.l36.best.sam

The parameters included in the sample command are:

• -l/--seedlen <int> seed length for -n (default: 28) -- Set to longest possible length of high-quality bases. Use the FastQC output to determine length of high-quality positions.
• -n/--seedmms <int> max mismatches in seed (can be 0-3, default: -n 2)
• -k <int> report up to <int> good alignments per read (default: 1) -- If you want only uniquely mapped reads, however, also use '-m 1' to ignore multi-mapped reads
• --solexa-quals (if input quals are from GA Pipeline ver. < 1.3) See the table at the top of FastQC output to identify the "encoding" scale
  
• --solexa1.3-quals or --phred64-quals (if input quals are from GA Pipeline ver. >= 1.3 and before Illumina 1.8) See the table at the top of FastQC output to identify the "encoding" scale
  
• --best (in the case of multi-mapped reads, keep only the best hit(s))
• --sam to get SAM output format (which is the best format for downstream analysis)

To see other parameters log into tak and type bowtie

​bowtie version 2

Sample command:

bsub bowtie2 --phred64 -L 22 -N 1 -x /nfs/genomes/mouse_gp_jul_07_no_random/bowtie/mm9 s_7.txt -S s7_mm9.L22.N1.sam

The parameters included in the sample command are:

• -L <int> length of seed substrings; must be >3 and <32 (default=22)
• -N <int> max # mismatches in seed alignment; can be 0 or 1 (default=0)
• --phred64 (if input quals are from GA Pipeline ver. >= 1.3 and before Illumina 1.8) See the table at the top of FastQC output to identify the "encoding" scale
  
• -S name of SAM output file

bowtie2 can also perform local alignments where the unaligned end(s) of a read are clipped (so, for example, remaining adapter won't prevent alignment) by adding the argument --local.

Other tools

Many other regular mapping tools are also available, although they generally require a tool-specific indexed version of the genome.

Splice-aware mappers

These mappers permit the beginning and end of a read to map to (originate from) different places in the genome, which is common for spliced RNA.

​tophat version 1

Running TopHat version 1 requires a change to a user's environment on tak (and only applies to the specific tak session. First run this command:

export PATH="/usr/local/share/tophat1:$PATH"

and then check that your terminal will use the correct TopHat version:

tophat --version

Sample command:

bsub tophat -o s_7_tophat_out --phred64-quals --no-novel-juncs --segment-length 20 -G /nfs/genomes/mouse_gp_jul_07_no_random/gtf/Mus_musculus.NCBIM37.67_noNT.gtf /nfs/genomes/mouse_gp_jul_07_no_random/bowtie/mm9 s_7.txt

The parameters included in the sample command are:

• -o/--output-dir <word> All output files will be created in this directory (default = tophat_out)
• --solexa-quals (if input quals are from GA Pipeline ver. < 1.3) See the table at the top of FastQC output to identify the "encoding" scale
  
• --phred64-quals or solexa1.3-quals (if input quals are from GA Pipeline ver. >= 1.3 before Illumina 1.8) See the table at the top of FastQC output to identify the "encoding" scale