== 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 [http://bowtie-bio.sourceforge.net/bowtie2/faq.shtml How is Bowtie 2 different from Bowtie 1?]

'''[http://bowtie-bio.sourceforge.net/index.shtml 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
}}}

Parameters included in the sample command:
  * '''-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
  * '''--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)
Choices for fastq encoding (which is listed as "Encoding" in the top "Basic Statistics" table of the FastQC output file).  See the [http://en.wikipedia.org/wiki/FASTQ_format FASTQ format page] for more details.
  * '''--solexa-quals'''         (for input quality scores from Illumina versions 1.2 and earlier)
  * '''--solexa1.3-quals''' or '''--phred64-quals'''     (for input quality scores from Illumina versions 1.3-1.7)
  * '''--phred33-quals'''         (default "Sanger format"; for input quality scores from Illumina versions 1.8 and later)

To see other parameters log into tak and type '''bowtie''' 


'''[http://bowtie-bio.sourceforge.net/bowtie2/index.shtml 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:
  * '''-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)
  * '''-S'''           name of SAM output file
Choices for fastq encoding (which is listed as "Encoding" in the top "Basic Statistics" table of the FastQC output file).  See the [http://en.wikipedia.org/wiki/FASTQ_format FASTQ format page] for more details.
  * '''--solexa-quals'''         (for input quality scores from Illumina versions 1.2 and earlier)
  * '''--phred64'''     (for input quality scores from Illumina versions 1.3-1.7)
  * '''--phred33'''         (default "Sanger format"; for input quality scores from Illumina versions 1.8 and later)

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.

'''[http://tophat.cbcb.umd.edu/ 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 --segment-length 20 -G /nfs/genomes/mouse_gp_jul_07_no_random/gtf/Mus_musculus.NCBIM37.67_noNT.gtf --no-novel-juncs /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)
  * '''--segment-length <int>'''  Shortest length of a spliced read that can map to one side of the junction.  For reads shorter than ~45 nt, set this to half the read length (so set '--segment-length 20' for 40-nt reads).  For longer reads, the default length (25) can be used.
  * '''-G <GTF file>''' Supply bowtie with a GTF file of transcript models.  This can help bowtie identify functions that may otherwise be missed.
  * '''--no-novel-juncs ''' Only look for spliced reads across junctions in the supplied GTF file.  Typically not used.
Choices for fastq encoding (which is listed as "Encoding" in the top "Basic Statistics" table of the FastQC output file).  See the [http://en.wikipedia.org/wiki/FASTQ_format FASTQ format page] for more details.
  * '''--solexa-quals'''         (for input quality scores from Illumina versions 1.2 and earlier)
  * '''--solexa1.3-quals''' or '''--phred64-quals'''     (for input quality scores from Illumina versions 1.3-1.7)

'''[http://tophat.cbcb.umd.edu/ tophat version 2]'''

TopHat version 2 uses bowtie2, rather than bowtie, for its mapping.

Sample command:
{{{
bsub tophat -o s_7_tophat_out --phred64-quals --segment-length 20 -G /nfs/genomes/mouse_gp_jul_07_no_random/gtf/Mus_musculus.NCBIM37.67_noNT.gtf --no-novel-juncs /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)
  * '''--segment-length <int>'''  Shortest length of a spliced read that can map to one side of the junction.  For reads shorter than ~45 nt, set this to half the read length (so set '--segment-length 20' for 40-nt reads).  For longer reads, the default length (25) can be used.
  * '''-G <GTF file>''' Supply bowtie with a GTF file of transcript models.  This can help bowtie identify functions that may otherwise be missed.
  * '''--no-novel-juncs ''' Only look for spliced reads across junctions in the supplied GTF file.  Typically not used.

Choices for fastq encoding (which is listed as "Encoding" in the top "Basic Statistics" table of the FastQC output file).  See the [http://en.wikipedia.org/wiki/FASTQ_format FASTQ format page] for more details.
  * '''--solexa-quals'''         (for input quality scores from Illumina versions 1.2 and earlier)
  * '''--solexa1.3-quals''' or '''--phred64-quals'''     (for input quality scores from Illumina versions 1.3-1.7)