Page Comparison

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We'll set up a new directory to perform the V. cholerae data alignment. But first make sure you have the FASTQ file to align and the vibCho bowtie2 index:

# Get the FASTQ to align
mkdir -p $SCRATCH/core_ngs/alignment/fastq
cp $CORENGS/alignment/*fastq.gz $SCRATCH/core_ngs/alignment/fastq/

# Set up the bowtie2 index
mkdir -p $SCRATCH/core_ngs/references/bt2/vibCho
cp $CORENGS/idx/bt2/vibCho/*.*  $SCRATCH/core_ngs/references/bt2/vibCho/

Make sure you're in an idev session with the bowtie2 BioContainers module loaded:

idev -m 120 -A OTH21164  -N 1 -r CoreNGSday4
module load biocontainers
module load bowtiebowtie2

Now set up a directory to do this alignment, with symbolic links to the bowtie2 index directory and the directory containing the FASTQ to align:

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• -x  vibCho/vibCho.O395.fa – prefix path of index files
• -U fq/cholera_rnaseq.fastq.gz – FASTQ file for single-end (Unpaired) alignment
• -S cholera_rnaseq.sam – tells bowtie2 to report alignments in SAM format to the specified file
• 2>&1 redirects standard error to standard output
  • while the alignment data is being written to the cholera_rnaseq.sam file, bowtie2 will report its progress to standard error.
• | tee aln.log takes the bowtie2 progress output and pipes it to the tee program
  • tee takes its standard input and writes it to the specified file and also to standard output
  • that way, you can see the progress output now, but also save it to review later (or supply to MultiQC)

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89006 reads; of these:
  89006 (100.00%) were unpaired; of these:
    206755902 (236.23%63%) aligned 0 times
    3822651483 (4257.95%84%) aligned exactly 1 time
    3010531621 (3335.82%53%) aligned >1 times
7693.77%37% overall alignment rate

When the job is complete you should have a cholera_rnaseq.sam file that you can examine using whatever commands you like.  Remember, to further process it downstream, you should create a sorted, indexed BAM file from this SAM output.

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module load samtools
cd $SCRATCH/core_ngs/alignment/vibCho
bowtie2 --local -x vibCho/vibCho.O395 -U fq/cholera_rnaseq.fastq.gz 2>aln_local.log | \
  samtools view -b > cholera_rnaseq.local.bam

89006 reads; of these:
  89006 (100.00%) were unpaired; of these:
    2706113359 (3015.40%01%) aligned 0 times
    3382846173 (3851.01%88%) aligned exactly 1 time
    2811729474 (3133.59%11%) aligned >1 times
6984.60%99% overall alignment rate

Exercise #5: BWA-MEM - Human mRNA-seq

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