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<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01//EN">
<html><head><title>Predicting Genes with AUGUSTUS</title>
<link rel="stylesheet" type="text/css" href="augustus.css">
<script src="tutorial.js" type="text/javascript"></script>
</head><body>
<font size=-1>
Navigate to <a href="index.html">main AugCGP tutorial</a>.
<a href="cactus.html">Cactus alignments and assembly Hubs</a>.
<a href="de_novo.html">AugCGP de novo</a>.
<a href="liftover.html">Annotation transfer with AugCGP</a>.
<a href="rnaseq+liftover.html">Combining RNA-Seq and annotation evidence</a>.
<a href="hgm.html">Cross-species consistency of gene sets</a>.
</font>
<div align="right">Show <a href="javascript:allOn()">all</a> / <a href="javascript:allOff()">no</a> details.</div>
<h1>RNA-Seq-based prediction with Augustus-cgp</h1>
This tutorial describes how RNA-Seq data can be incorporated into Augustus-cgp.
In general, the same types of extrinsic evidence can be incorporated as in single-species gene finding with Augustus (including RNA-Seq, cDNA, ESTs, protein sequences, etc). In cgp mode, evidence is species-specific an can be incorporated for each or a subset of genomes.
<h2 id="rnaseq2hints">1. Generate hints from RNA-Seq</h2>
For most species raw RNA-Seq data (in fastq format) is readly available and can be downloaded
from the <a href="http://www.ncbi.nlm.nih.gov/sra">NCBI Sequence Read Archive (SRA)</a>.<br><br>
<a href="javascript:onoff('tp')" class="dlink"><span id="tp" title="tpd" class="dcross">[+]</span>
<span class="dtitle">Some advice on how to choose the RNA-Seq libraries...</span></a> <br>
<div id="tpd" class="details" style="display:none;">
<ul>
<li> whenever possible, choose paired-end RNA-Seq reads</li>
<li> pay attention to the timeliness of the data. More recent studies generally use
better sequencing technologies and can produce longer reads.<br>
A read length of at least 2x100 bps is recommended.</li>
<li> check in the protocol, how rRNA is removed. Rather use Poly(A) tagged libraries than
Ribo-Zero depleted libraries, as the latter contain all kinds of non-coding elements and may also
have a high coverage in introns and intergenic regions.</li>
</ul>
</div>
<br>
In order to integrate RNA-Seq into Augustus, the usual procedure involves
<ul>
<li> aligning RNA-Seq reads to the source genome with a spliced-aligner (f.e. <a href="https://github.com/alexdobin/STAR">STAR</a>)</li>
<li> quality and uniqueness filtering of alignments with <tt>filterBam</tt></li>
<li> generation of intron hints with <tt>bam2hints</tt></li>
<li> generation of exonpart hints with <tt>bam2wig</tt> and <tt>wig2hints.pl</tt></li>
</ul>
We will skip this procedure as it is explained in detail in other tutorials (see section 'RNA-Seq integration' in the <a href="http://bioinf.uni-greifswald.de/bioinf/wiki/pmwiki.php?n=Augustus.Augustus">Augustus Wiki</a>)<br>
and assume that we have generated <a href="data/hints/" >hints files</a> for some of the species (chicken, human, mouse and rhesus) in our clade.
<h2 id="loadRNASeq">2. Load RNA-Seq hints into the SQLite database</h2>
<span class="assignment">Prepare</span> a text file with a list of species names and location of
the corresponding hints files.<br><br>
<a href="javascript:onoff('cft')" class="dlink"><span id="cft" title="cftd" class="dcross">[+]</span>
<span class="dtitle">file format...</span></a> <br>
<div id="cftd" class="details" style="display:none;">
<pre class="code">
name_of_genome_1 path/to/hintsfile_1
name_of_genome_2 path/to/hintsfile_2
...
name_of_genome_N path/to/hintsfile_N
</pre>
</div>
</br>
<pre class="code">
for f in $PWD/hints/*.gff; do echo -ne "$(basename $f .hints.gff)\t$f\n"; done >hints.tbl
</pre>
The file <span class="result"><tt>hints.tbl</tt></span> will now look like this (except for the parent directory)<br>
<pre class="code">
galGal4 /var/www/augustus/htdocs/binaries/tutorial-cgp/data/hints/galGal4.hints.gff
hg38 /var/www/augustus/htdocs/binaries/tutorial-cgp/data/hints/hg38.hints.gff
mm10 /var/www/augustus/htdocs/binaries/tutorial-cgp/data/hints/mm10.hints.gff
rheMac3 /var/www/augustus/htdocs/binaries/tutorial-cgp/data/hints/rheMac3.hints.gff
</pre>
If you don't have a database with the genomes, yet, <span class="assignment">follow</span> the instructions in
<a href="de_novo.html#loadFa">1. Load genomes ...</a><br>
to create the database <tt>vertebrates.db</tt>.<br><br>
<span class="assignment">Make</span> a copy of the database
<pre class="code">
cp vertebrates.db vertebrates_rnaseq.db
</pre>
and <span class="assignment">load</span> the hints into the new database
<pre class="code">
while read line
do
species=$(echo "$line" | cut -f 1)
hints=$(echo "$line" | cut -f 2)
load2sqlitedb --noIdx --species=$species --dbaccess=vertebrates_rnaseq.db $hints
done <hints.tbl
</pre>
<span class="assignment">Finalize</span> database by creating indices on the tables
<pre class="code">
load2sqlitedb --makeIdx --dbaccess=vertebrates_rnaseq.db
</pre>
You can <span class="assignment">check</span> if loading was successful with following
database query
<pre class="code">
sqlite3 -header -column vertebrates_rnaseq.db "\
SELECT count(*) AS '#hints',typename,speciesname\
FROM (hints as H join featuretypes as F on H.type=F.typeid)\
natural join speciesnames\
GROUP BY speciesid,typename;"
</pre>
that returns a summary of how many hints of each type are in the database for each species.
<pre class="code">
#hints typename speciesname
---------- ---------- -----------
3368 exonpart galGal4
129 intron galGal4
7905 exonpart hg38
267 intron hg38
7930 exonpart mm10
378 intron mm10
11050 exonpart rheMac3
265 intron rheMac3
</pre>
<h2 id="extrinsic">3. Prepare an extrinsic config file</h2>
Start by <span class="assignment">copying</span> following extrinsic configuration file:
<pre class="code">
cp ${AUGUSTUS_CONFIG_PATH}/extrinsic/extrinsic-cgp.cfg extrinsic-rnaseq.cfg
</pre>
<span class="assignment">Open</span> the <tt>extrinsic-rnaseq.cfg</tt> file with a text editor,
<span class="assignment">go</span> to the first <tt>[GROUP]</tt> section and replace the next line
<pre class="code">
[GROUP] # replace 'none' by the names of genomes with src=W and src=E hints in the database
none
</pre>
as instruced by the names of genomes with RNA-Seq hints, i.e.
<pre class="code">
[GROUP] # replace 'none' by the names of genomes with src=W and src=E hints in the database
hg38 mm10 rheMac3 galGal4
</pre>
<a href="javascript:onoff('cfg')" class="dlink"><span id="cfg" title="cfgd" class="dcross">[+]</span>
<span class="dtitle">format of the extrinsic.cfg file in cgp mode ...</span></a> <br>
<div id="cfgd" class="details" style="display:none;">
In cgp mode hints can be integrated for multiple species.
In order to have different extrinsic config settings for different species,
multiple <tt>[GENERAL]</tt> tables are specified. Each table is followed by a <tt>[GROUP]</tt> section,
a single line, in which a subset of the species is listed, for which the table is valid.
Use the same species identifiers as in the genome alignment and in the phylogenetic tree.
If a species is not assigned to any of the tables, all hints for that species are
ignored. To assign all species to a single table, the key 'all' can be used instead of itemizing
every single species identifier. Use the key 'other' to specify a table for all species, not
listed in any previous table.
Note that the source RM must be specified in case that the softmasking option is turned on.
Also note that all tables have the same dimension, i.e. each table must contain all sources
listed in the section <tt>[SOURCES]</tt>, even sources for which no hints exist for any of species
in group.
</div>
</br>
<h2 id="runAug">4. Run AUGUSTUS-CGP with RNA-Seq hints</h2>
<span class="assignment">Create</span> a new folder for the rnaseq experiments and
<span class="assignment">switch</span> to the new directory
<pre class="code">
mkdir augCGP_rnaseq
cd augCGP_rnaseq
</pre>
For convenience <span class="assignment">assign</span> each alignment chunk to a job ID by
creating softlinks
<pre class="code">
num=1
for f in ../mafs/*.maf; do ln -s $f $num.maf; ((num++)); done
</pre>
<span class="assignment">Run</span> Augustus with retrieval of hints from the
database (~3min).<br>
<pre class="code">
for ali in *.maf
do
id=${ali%.maf} # remove .maf suffix
augustus \
--species=human \
--softmasking=1 \
--treefile=../tree.nwk \
--alnfile=$ali \
--dbaccess=../vertebrates_rnaseq.db \
--speciesfilenames=../genomes.tbl \
--alternatives-from-evidence=0 \
--dbhints=1 \
--UTR=1 \
--allow_hinted_splicesites=atac \
--extrinsicCfgFile=../extrinsic-rnaseq.cfg \
--/CompPred/outdir=pred$id > aug$id.out 2> err$id.out &
done
</pre>
The option <tt>UTR=1</tt> enables the UTR model and is recommended whenever 'exonpart' hints are incorporated.<br><br>
This will generate the folders <span class="result"><tt>pred*/</tt></span> (one for each alignment chunk)
that contain gff files with gene predictions for each input genome.
<pre class="code">
bosTau8.cgp.gff
canFam3.cgp.gff
galGal4.cgp.gff
hg38.cgp.gff
mm10.cgp.gff
monDom5.cgp.gff
rheMac3.cgp.gff
rn6.cgp.gff
</pre>
Note that the parallelization with the bash '&' command above is quite simple and rather for demonstration purposes.<br>
For real applications with several hundreds or thousands of alignment chunks, we recommend to
run job arrays on a compute cluster.
<h2 id="abinitiopred"><a href="de_novo.html#merge">5. Merge gene predictions from parallel runs</a></h2>
<h2 id="makeBeds">6. Upload gene predictions into the assembly hub</h2>
<span class="assignment">Convert</span> the final gene predictions from gff to BED format and place
each BED file in a separate folder with the name of the corresponding genome. It is important that directory names are consistent with the names in the HAL alignment.
<pre class="code">
for f in joined_pred/*.gff
do
mkdir "$(basename $f .gff)"
gtf2bed.pl <$f >$(basename $f .gff)/augCGP_rnaseq.bed --itemRgb=34,139,34
done
</pre>
Specify any RGB color you like for the track with option <tt>--itemRgb</tt>, e.g. <span style="color:rgb(34,139,34);">34,139,34.</span><br>
The name of the current directory (i.e. <tt>augCGP_rnaseq</tt>) will be used as track name on the browser.<br>
<span class="assignment">Switch</span> back to the main working directory <tt>data/</tt>
<pre class="code">
cd ..
</pre>
and rerun the <tt>hal2assemblyHub.py</tt> script. Include gene tracks with option <tt>--bedDirs</tt>
<pre class="code">
hal2assemblyHub.py vertebrates.hal vertHub --lod \
--alignability --gcContent \
--hub vertCompHub --shortLabel VertebratesCompHub \
--bedDirs augCGP_rnaseq \
--tabBed \
--maxThreads=10 --longLabel "Vertebrates Comparative Assembly Hub"
</pre>
You can also include gene tracks from other exercises by passing a comma-separated list of directories e.g.
<tt>--bedDirs refseq,augCGP_denovo,augCGP_rnaseq,augCGP_liftover,...</tt><br><br>
<span class="assignment">Repeat</span> <a href="cactus.html#loadHub">4. Load the hub and browser the alignment</a>.<br>
</body></html>
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