NAME

       htseq-qa - Perform simple quality assesment of high-throughput sequencing reads

       The  Python  script  htseq-qa  takes  a  file with sequencing reads (either raw or aligned
       reads) and produces a PDF file with useful plots to assess the technical quality of a run.

PLOT

       Here is a typical plot: [image]

       The plot is made from a SAM file, which contained aligned and unalignable reads.  The left
       column  is  made from the non-aligned, the right column from the aligned reads. The header
       informs you about the name of the SAM file, and the number of reads.

       The upper row shows how often which base was called for each position in the read. In this
       sample, the non-alignable reads have a clear excess in A. The aligned reads have a balance
       between complementing reads: A and C (reddish colours) have equal levels, and so do C  and
       G  (greenish  colours).  The sequences seem to be AT rich. Furthermore, nearly all aligned
       reads start with a T, followed by an A, and then, a C in 70% and an A in 30% of the reads.
       Such  an  imbalance  would  be reason for concern if it has no good explanation. Here, the
       reason is that the fragmentation of the sample was done by enzyme digestion.

       The lower half shows the abundance of base-call quality scores at the different  positions
       in  the read. Nearly all aligned reads have a quality of 34 over their whole length, while
       for the non-aligned reads, some reads have lower quality scores towards their ends.

USAGE

       Note that htseq-qa needs matplotlib to produce the plot,  so  you  need  to  install  this
       module, as described here on the matplotlib web site.

       After you have installed HTSeq (see install) and matplotlib, you can run htseq-qa from the
       command line:

          htseq-qa [options] read_file

       If the file htseq-qa is not in your path, you can, alternatively, call the script with

          python -m HTSeq.scripts.qa [options] read_file

       The read_file is either a FASTQ file or a SAM file. For  a  SAM  file,  a  plot  with  two
       columns is produced as above, for a FASTQ file, you get only one column.

       The  output  is  written into a file with the same name as read_file, with the suffix .pdf
       added. View it with a PDF viewer such as the Acrobat Reader.

   Options
       -t <type>, --type=<type>
              The file type of the read_file. Supported values for <type> are:

              · sam: a SAM file (Note that the SAMtools contain  Perl  scripts  to  convert  most
                alignment formats to SAM)

              · solexa-export:  an  _export.txt  file  as produced by the SolexaPipeline software
                after aligning with Eland (htseq-qa expects the new Solexa  quality  encoding  as
                produced by version 1.3 or newer of the SolexaPipeline)

              · fastq: a FASTQ file with standard (Sanger or Phred) quality encoding

              · solexa-fastq:  a  FASTQ  file  with  Solexa  quality encoding, as produced by the
                SolexaPipeline after base-calling with Bustard (htseq-qa expects the  new  Solexa
                quality encoding as produced by version 1.3 or newer of the SolexaPipeline)

       -o <outfile>, --outfile=<outfile>
              output filename (default is <read_file>``.pdf``)

       -r <readlen>, --readlength=<readlen>
              the maximum read length (when not specified, the script guesses from the file

       -g <gamma>, --gamma=<gamma>
              the gamma factor for the contrast adjustment of the quality score plot

       -n, --nosplit
              do not split reads in unaligned and aligned ones, i.e., produce a one-column plot

       -m, --maxqual
              the maximum quality score that appears in the data (default: 40)

       -h, --help
              Show a usage summary and exit

AUTHOR

       Simon Anders

COPYRIGHT

       2010, Simon Anders