# Align and count gene features¶

Several aligners exist for mapping reads back to a reference genome (Bowtie, Bowtie2, BWA, SOAP, TopHat, etc.). Each aligner was developed for a specific use case in mind and one should be aware of these differences. For example, TopHat (Bowtie) was optimized for identification of splice junctions. We don’t have to worry about that for microbes, but the aligner itself (Bowtie) works just fine even in gene dense genomes like bacteria. Another caveat is there are two flavors of Bowtie: Bowtie1 and Bowtie2. Bowtie1 seems to perform better for 50 bp reads while 100+ bp reads tend to be faster using Bowtie2.

But what about the other aligners? Well, to start, look and see what other people are using in the literature, talk to your colleagues, or your friendly neighborhood bioinformatician.

The take home messages are:

• Many of these tools have great documentation, so it’s worth having a look to see if they explicitly state the use case in mind and whether your experiment/data fits.
• Check the literature
• Try them out and compare the results

The next component in all of this is whether you are doing reference or reference free assembly. More on reference free assembly later. For our purposes, we have a reference.

My favorite place to download a reference genome and annotation files are from Ensembl.

## Align the trimmed reads with Bowtie¶

cd ~/TranscriptomicsWorkshop/Alignment
wget http://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/microbial-2015-09-24/MtbCDC1551.fa


Build the index of the genome for bowtie to use to perform the mapping/alignment process:

bowtie-build MtbCDC1551.fa MtbCDC1551


The counting transcript abundance step won’t read in compressed files (files that end with .gz), so we will unzip them now and hand them to bowtie (the aligner) unzipped.

We could do this in individual lines by typing something like:

# (don't run this!)
gunzip -c gly7a.fq.gz > gly7a.fq


But this would be rather tedious to do this for every file. Then we have to move the files to our Alignment folder by typing something like:

# (don't run this!)
mv gly7a.fq ~/TranscriptomicsWorkshop/Alignment


But again, this would be rather tedious to do... So what we will do is script it and combine the decompressing with moving the file into a single command with &&. This is really useful in a Linux type system to string two commands together. What it does is say “if the command to the left of the && works, then do the command to the right of the &&.” Finally, the . in the move (mv) command means “the folder you are in right now”.

So, let’s do it!

Unzip the files and move the files to the Alignment folder (which we should already be in). Copy and paste the lines below into the terminal:

#decompress the files
for compfiles in ../QC/Trimmomatic/*.fq.gz
do
FILENAME=${compfiles%.*.*} gunzip -c$FILENAME.fq.gz > $FILENAME.fq && mv$FILENAME.fq .
done


Now, we have been adding prefixes to files so we have an idea based on the name, how the file has been modified during the analysis. We want to strip off this prefix before moving on so that we can add a new one. To do this, we can use the move (mv) command again to rename a file.

The syntax looks something like this as an example:

# (don't run this!)
mv trimmedgly7a.fq gly7a.fq


So essentially, you have your command, mv and then the file you want to rename and then the new name of the file.

But to do this for every file is a pain and error prone. Script it!:

#rename the files by stripping off the trimmed prefix
for renametrim in *.fq
do
LESSPREFIX=$(basename${renametrim//trimmed/})
mv $renametrim$LESSPREFIX
done


Whew, that was a lot of work to get to the alignment step. We are going to use Bowtie to do the alignment of the reads. Bowtie has a lot of options to tailor the alignment procedure. Here we only specifiy the -S option to let Bowtie know that we want our output in .sam format. The general syntax for running bowtie is as follows:

# (don't run this!)
bowtie -S [index file prefix from the bowtie-build command] [your input file] > [your output file that ends in .sam]


We have another new thing that looks like the greater than (>) sign. This says “take the output from the command on the left and put it in a new file on the right”.

#align the reads
for alignment in *.fq
do
FILENAME=$(basename${alignment%.*})
PREFIX=align
NEWALIGNFILE=${PREFIX}${FILENAME}
bowtie -S MtbCDC1551 $FILENAME.fq >$NEWALIGNFILE.sam
done

#clean up the FASTQ files
rm *.fq


## Count gene features/quantify transcript abundance with HTSeq¶

So now that we have aligned the reads to the genome, we want to count transcript abundance per gene using an annotation file (MtbCDC1551.gtf) that was generated when the genome is annotated. This file tells the software the gene coordinates and strandedness.

cd ../TranscriptAbund
wget http://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/microbial-2015-09-24/MtbCDC1551.gtf


Let’s have a look at what the first few lines of this file look like:

head MtbCDC1551.gtf


It should look something like this:

Chromosome  protein_coding  exon    1       1524    .       +       .        gene_id "MT0001"; transcript_id "AAK44224"; exon_number "1"; gene_name "dnaA"; transcript_name "dnaA/AAK44224"; seqedit "false";
Chromosome  protein_coding  CDS     1       1521    .       +       0        gene_id "MT0001"; transcript_id "AAK44224"; exon_number "1"; gene_name "dnaA"; transcript_name "dnaA/AAK44224"; protein_id "AAK44224";
.
.
.


We can see the entire exon, its coordinates, the strand, gene_id, gene_name, etc. The software we will use (HTSeq) requires this file to “know” how to interpret the alignment file to count whether a transcript was observed.

We need to strip off prefixes again and we encounter another new command called copy (cp). The general syntax is similar to move (mv):

# (don't run this!)
cp [the file you want to copy] [where you want to copy the file to]


So let’s take off the prefixes and copy the stripped files to the TranscriptAbund folder:

#strip off the align prefix and move the files into the TranscriptAbund folder
for renamealign in ../Alignment/*.sam
do
LESSPREFIX=$(basename${renamealign//align/})
cp renamealign ../TranscriptAbund/LESSPREFIX
done


HTSeq has a spectacular function called htseq-count. This function will quantify transcript abundances for us. It also has several options we can specify, with two particularly important ones that tell it how to call whether a read is within a coding region (-m) and whether our data is stranded (–stranded). Have a look at the documentation on the three choices for the -m option. Further, if you are doing a single-end RNA-seq experiment with the Illumina TruSeq library preparation kit, your data will be reverse stranded. You can experiment with this to see by specifying –stranded=forward or –stranded=no.

The syntax is as follows:

htseq-count -m [how you want HTSeq to call a read in a gene region] --stranded=[reverse, forward, no] [alignment file that ends in .sam] [genome annotation file that ends with .gtf] > [your output file that ends in .counts]


Let’s count!

To run the software:

for counts in *.sam
do
FILENAME=$(basename${counts%.*})
PREFIX=abundcount_
NEWMAPFILE=${PREFIX}${FILENAME}
htseq-count -m intersection-nonempty --stranded=reverse $FILENAME.sam MtbCDC1551.gtf >$NEWMAPFILE.counts
done

#clean up the .sam files
rm *.sam


Congratulations! We are now ready to do differential gene expression.

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