Page Comparison

Tip

title	Reservations

Use our summer school reservation (CoreNGS

-Thu

) when submitting batch jobs to get higher priority on the ls6 normal queue

today:sbatch --reservation=CoreNGS-Thu <batch_file>.slurm

.

Code Block

language	bash
title	Request an interactive (idev) node

# Request a 180 minute interactive node on the normal queue using our reservation
idev -m 120 -N 1 -A OTH21164 -r CoreNGS
idev -m 120 -N 1 -A TRA23004 -r CoreNGS

# Request a 120 minute idev node on the development queue 
idev -m 120 -N 1 -A OTH21164 -p development
idev -m 120 -N 1 -A TRA23004 -p development

Code Block

language	bash
title	Submit a batch job

# Using our reservation
sbatch --reseservation=CoreNGS <batch_file>.slurm

Note that the reservation name (CoreNGS) is different from the TACC allocation/project for this class, which is OTH21164.

Table of Contents

Exercise #3: PE alignment with BioITeam scripts

...

Code Block

language	bash

align_bwa_illumina.sh 2022_0506_0510
Align Illumina SE or PE data with bwa. Produces a sorted, indexed,
duplicate-marked BAM file and various statistics files. Usage:

align_bwa_illumina.sh <aln_mode> <in_file> <out_pfx> <assembly> [ paired trim_sz trim_sz2 seq_fmt qual_fmt ]

Required arguments:
  aln_mode  Alignment mode, either global (bwa aln) or local (bwa mem).
  in_file   For single-end alignments, path to input sequence file.
            For paired-end alignments using fastq, path to the the R1
            fastq file which must contain the string 'R1' in its name.
            The corresponding 'R2' must have the same path except for 'R1'.
  out_pfx   Desired prefix of output files in the current directory.
  assembly  One of hg38, hg19, hg38, mm10, mm9, sacCer3, sacCer1, ce11, ce10,
            danRer7, hs_mirbase, mm_mirbase, or reference index prefix.
Optional arguments:
  paired    0 = single end alignment (default); 1 = paired end.
  trim_sz   Size to trim reads to. Default 0 (no trimming)
  trim_sz2  Size to trim R2 reads to for paired end alignments.
            Defaults to trim_sz
  seq_fmt   Format of sequence file (fastq, bam or scarf). Default is
            fastq if the input file has a '.fastq' extension; scarf
            if it has a '.sequence.txt' extension.
  qual_type Type of read quality scores (sanger, illumina or solexa).
            Default is sanger for fastq, illumina for scarf.
Environment variables:
  show_only  1 = only show what would be done (default not set)
  aln_args   other bowtie2 options (e.g. '-T 20' for mem, '-l 20' for aln)
  no_markdup 1 = don't mark duplicates (default 0, mark duplicates)
  run_fastqc 1 = run fastqc (default 0, don't run). Note that output
             will be in the directory containing the fastq files.
  keep       1 = keep unsorted BAM (default 0, don't keep)
  bwa_bin    BWA binary to use. Default bwa 0.7.x. Note that bwa 0.6.2
             or earlier should be used for scarf and other short reads.
  also: NUM_THREADS, BAM_SORT_MEM, SORT_THREADS, JAVA_MEM_ARG

Examples:
  align_bwa_illumina.sh local  ABC_L001_R1.fastq.gz my_abc hg38 1
  align_bwa_illumina.sh global ABC_L001_R1.fastq.gz my_abc hg38 1 50
  align_bwa_illumina.sh global sequence.txt old sacCer3 0 '' '' scarf solexa

...

Code Block

language	bash
title	Run multiple alignments using the TACC batch system

# Make sure you're not in an idev session by looking at the hostname
hostname
# If the hostname looks like "c455-004.ls6.tacc.utexas.edu", exit the idev session

# Copy over the Yeast data if needed
mkdir -p $SCRATCH/core_ngs/alignment/fastq
cp $CORENGS/alignment/Sample_Yeast*.gz $SCRATCH/core_ngs/alignment/fastq/

# Make a new alignment directory for running these scripts
mkdir -p $SCRATCH/core_ngs/alignment/bwa_script
cd $SCRATCH/core_ngs/alignment/bwa_script
ln -ssf -f ../fastq

# Copy the alignment commands file and submit the batch job
cd $SCRATCH/core_ngs/alignment/bwa_script 
cp $CORENGS/tacc/aln_script.cmds .

# Use -a TRA23004 below if OTH21164 hasn't been working for you...
launcher_creator.py -j aln_script.cmds -n aln_script -t 01:00:00 -w 4 -a OTH21164 -q normal
sbatch --reservation=CoreNGS-Thu aln_script.slurm 

# or
launcher_creator.py -j aln_script.cmds -n aln_script -t 01:00:00 -w 4 -a OTH21164 -q development
sbatch aln_script.slurm  

showq -u

...

Since this was a paired end alignment there is paired-end specific information reportedinformation reported. Note that this Yeast dataset was of poor quality, especially the R2 reads. You can see this by the relatively low R1 alignment rate (~63%) and the really low R2 alignment rate (~28%).

You can also view statistics on insert sizes for properly paired reads in the bwa_global.iszinfo.txt file. This tells you the average (mean) insert size, standard deviation, mode (most common value), and fivenum values (minimum, 1st quartile, median, 3rd quartile, maximum).

...

In this exercise, we will use some RNA-seq data from Vibrio cholerae, published on GEO here, and align it to a reference genome.

...

Code Block

language	bash
title	Start an idev session

idev -m 120 -A OTH21164 -N 1 -r CoreNGS-Thu      # or -A TRA23004 
# or
idev -m 90 -A OTH21164 -N 1 -p development   # or -A TRA23004

Go ahead and load the bowtie2 module so we can examine some help pages and options.

...

Code Block

language	bash

idev -m 120 -A OTH21164  -N 1 -r CoreNGS-Thu    # or -A TRA23004
# or
idev -m 90 -A OTH21164 -N 1 -p development  # or -A TRA23004

module load biocontainers
module load bowtie2

...

Expand

title	Answer

Code Block

language	bash

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

Reports these alignment statistics:

Code Block
89006 reads; of these: 89006 (100.00%) were unpaired; of these: 13359 (15.01%) aligned 0 times 46173 (51.88%) aligned exactly 1 time 29474 (33.11%) aligned >1 times 84.99% overall alignment rate

Interestingly, the local alignment rate here is lower than we saw with the global alignment. Usually local alignments have higher alignment rates than corresponding global ones.

...

Using bwa mem for RNA-seq alignment is sort of a "poor man's" RNA-seq alignment method. Real splice-aware aligners like tophat2STAR, hisat2 or STAR tophat have more complex algorithms (as shown below) – and take a lot more time!

...

BWA MEM does not know about the exon structure of the genome. But it can align different sub-sections of a read to two different locations, producing two alignment records from one input read (one . One of the two will be marked as secondary (0x100 flag).

...

Code Block

language	bash
title	Run multiple alignments using the TACC batch system

# Make sure you're in an idev session
idev -m 120 -N 1 -A OTH21164 -r CoreNGS-Thu      # or -A TRA23004
# or
idev -m 90 -N 1 -A OTH21164 -p development   # or -A TRA23004

# Load the modules we'll need
module load biocontainers
module load bwa
module load samtools

# Copy over the FASTQ data if needed
mkdir -p $SCRATCH/core_ngs/alignment/fastq
cp $CORENGS/alignment/*.gz $SCRATCH/core_ngs/alignment/fastq/

# Make a new alignment directory for running these scripts
cds
mkdir -p core_ngs/alignment/bwamem
cd core_ngs/alignment/bwamem
ln -sf ../fastq
ln -sf /work/projects/BioITeam/ref_genome/bwa/bwtsw/hg38

Now take a look at bwa mem usage (type bwa mem with no arguments, or bwa mem 2>&1 | more). The most important parameters are the following:

...

Code Block

language	bash

cd $SCRATCH/core_ngs/alignment/bwamem
bwa mem -M hg38/hg38.fa fastq/human_rnaseq.fastq.gz 2>hs_rna.bwamem.log | \
  samtools view -b | \
  samtools sort -O BAM -T human_rnaseq.tmp > human_rnaseq.sort.bam

...

Versions Compared

Old Version 32

New Version Current

Key

Exercise #3: PE alignment with BioITeam scripts