HTSeq version in-consistency and the STRANDED option

[LeonDK]

Hi all,

I have used tophat2 to map human RNASeq reads to UCSC Hg19.
The result hereof is naturally a BAM-file. I now want to count reads mapped to genes and for this purpose I have chosen HTSeq.
I have downloaded and installed HTSeq-0.6.1, but when I type

htseq-count -h

It says

Public License v3. Part of the 'HTSeq' framework, version 0.5.4p5.

Is something wrong with the installation?

And also, what exactly does the '-s' option do?

  -s STRANDED, --stranded=STRANDED
                        whether the data is from a strand-specific assay.
                        Specify 'yes', 'no', or 'reverse' (default: yes).
                        'reverse' means 'yes' with reversed strand
                        interpretation

What does 'strand-specific assay' mean and should I use the '-s' option, when counting human RNASeq reads mapped to Hg19?

Cheers,
Leon

[dpryan]

Presumably it's finding the older version before the newer one. Just remove the old version.

Regarding strandedness, see here. For context, strand-specific is the same concept as directional. If you don't know what a strand is then ask a biologist for a primer on the central dogma of molecular biology.

[LeonDK]

Presumably it's finding the older version before the newer one. Just remove the old version.

Regarding strandedness, see here. For context, strand-specific is the same concept as directional. If you don't know what a strand is then ask a biologist for a primer on the central dogma of molecular biology.

Hi dpryan,

I am sure, that I am calling the htseq-count I actually installed...

With respect to the strand-specific assaying - I am a newbie to NGS, hence my profile and what may seem "simple" questions... In NGS context, I am not sure I understand exactly what it means, that a sequencing protocol is strand-specific?

Cheers,
Leon

[blancha]

Yes, RNA-Seq can be strand-specific.
In fact, at our molecular biology platform, we now only do strand-specific RNA-Seq, since it eliminates the ambiguity when there are different genes on opposite strands.
There are several protocols to prepare strand-specific RNA-Seq libraries. We use the dUTP protocol.

Regarding your problem with the htseq-count version, here are a 2 suggestions.

First, the correct htseq-count version will appear if it is properly installed.

[blancha@lg-1r17-n03 deduplicatebismark]$ htseq-count -h
Usage: htseq-count [options] alignment_file gff_file

This script takes an alignment file in SAM/BAM format and a feature file in
GFF format and calculates for each feature the number of reads mapping to it.
See http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details.

Options:
  -h, --help            show this help message and exit
  -f SAMTYPE, --format=SAMTYPE
                        type of <alignment_file> data, either 'sam' or 'bam'
                        (default: sam)
  -r ORDER, --order=ORDER
                        'pos' or 'name'. Sorting order of <alignment_file>
                        (default: name). Paired-end sequencing data must be
                        sorted either by position or by read name, and the
                        sorting order must be specified. Ignored for single-
                        end data.
  -s STRANDED, --stranded=STRANDED
                        whether the data is from a strand-specific assay.
                        Specify 'yes', 'no', or 'reverse' (default: yes).
                        'reverse' means 'yes' with reversed strand
                        interpretation
  -a MINAQUAL, --minaqual=MINAQUAL
                        skip all reads with alignment quality lower than the
                        given minimum value (default: 10)
  -t FEATURETYPE, --type=FEATURETYPE
                        feature type (3rd column in GFF file) to be used, all
                        features of other type are ignored (default, suitable
                        for Ensembl GTF files: exon)
  -i IDATTR, --idattr=IDATTR
                        GFF attribute to be used as feature ID (default,
                        suitable for Ensembl GTF files: gene_id)
  -m MODE, --mode=MODE  mode to handle reads overlapping more than one feature
                        (choices: union, intersection-strict, intersection-
                        nonempty; default: union)
  -o SAMOUT, --samout=SAMOUT
                        write out all SAM alignment records into an output SAM
                        file called SAMOUT, annotating each line with its
                        feature assignment (as an optional field with tag
                        'XF')
  -q, --quiet           suppress progress report

Written by Simon Anders ([email protected]), European Molecular Biology
Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General
Public License v3. Part of the 'HTSeq' framework, version 0.6.1p1.

Second, you should verify if you are calling the version you just installed

which htseq-count

Finally, and this is a common problem, verify that you have changed the PYTHONPATH variable to point to the updated HTSeq library.

echo $PYTHONPATH

[LeonDK]

Ok, so found out, that the RNASeq data I have is stranded / strand-specific, so that's all good...

Reg. HTSeq it seemed that the $PYTHONPATH needed updating

Cheers,
Leon