Page History

...

Stage the alignment data

First connect to login5stampede2.ls5.tacc.utexas.edu and start an idev session. This should be second nature by now

Code Block

language	bash
title	Start an idev session

idev -p development -m 120 -A UT-2015-05-18 -N 1 -n 68 --reservation=BIO_DATA_week_1

Then stage the sample datasets and references we will use.

Code Block

language	bash
title	Get the alignment exercises files

mkdir -p $SCRATCH/core_ngs/alignmentreferences/fastqfasta
mkdir -p $SCRATCH/core_ngs/referencesalignment/fastafastq
cp $CORENGS/alignmentreferences/*fastq.gzfa $SCRATCH/core_ngs/alignmentreferences/fastqfasta/
cp $CORENGS/referencesalignment/*fastq.fagz $SCRATCH/core_ngs/references/fasta//alignment/fastq/
cd $SCRATCH/core_ngs/alignment/fastq

These are descriptions of the FASTQ files we copied:

...

Code Block

language	bash
title	BWA hg19 index location

/workwork2/projects/BioITeam/ref_genome/bwa/bwtsw/hg19

Tip

The BioITeam maintains a set of reference indexes for many common organisms and aligners. They can be found in aligner-specific sub-directories of the /workwork2/projects/BioITeam/ref_genome area. E.g.:

Code Block

language	bash

/workwork2/projects/BioITeam/ref_genome/
   bowtie2/
   bwa/
   hisat2/
   kallisto/
   star/
   tophat/

...

Code Block

language	bash
title	grep to match contig names in a FASTA file

cd $SCRATCH/core_ngs/references/fasta
grep -P '^>' sacCer3.fa | more

...

We might be able to get away with just using this literal alone as our regex, specifying '>' as the command line argument. But for grep, the more specific the pattern, the better. So we constrain where the > can appear on the line. The special carat ( ^ ) character metacharacter represents "beginning of line". So ^> means "beginning of a line followed by a > character".

...

alignment type	aligner options	pro's	con's
global with bwa	SE: bwa aln <R1> bwa samse PE: bwa aln <R1> bwa aln <R2> bwa sampe	simple to use (take default options) good for basic global alignment	multiple steps needed
global with bowtie2	bowtie2 --global	extremely configurable can be used for RNAseq alignment (after adapter trimming) because of its many options	complex (many options)
local with bwa	bwa mem	simple to use (take default options) very fast no adapter trimming needed good for simple RNAseq analysis the secondary alignments it reports provide splice junction information	always produces alignments with secondary reads must be filtered if not desired
local with bowtie2	bowtie2 --local	extremely configurable no adapter trimming needed good for small RNA alignment because of its many options	complex – many options

...

Like other tools you've worked with so far, you first need to load bwa. Do that now, and then enter bwa with no arguments to view the top-level help page (many NGS tools will provide some help when called with no arguments). Note that bwa is available both from the standard TACC module system and as BioContainers. module.

Code Block

language	bash

module load bwa
bwa

Expand

title	Make sure you're in a idev session

Make sure you're in an idev session. If you're in an idev session, the hostname command will display a name like c455-021.stampede2.tacc.utexas.edu. But if you're on a login node the hostname will be something like login3.stampede2.tacc.utexas.edu.

If you're on a login node, start an idev session like this:

Code Block

language	bash
title	Start an idev session

idev -p development -m 120 -A UT-2015-05-18 -N 1 -n 68 --reservation=BIO_DATA_week_1

Code Block

language	bash

module load biocontainers  # takes a while
module load bwa
bwa

Code Block

title	BWA suite usage

Program: bwa (alignment via Burrows-Wheeler transformation)
Version: 0.7.17-r1188

Code Block

title	BWA suite usage

Program: bwa (alignment via Burrows-Wheeler transformation)
Version: 0.7.16a-r1181
Contact: Heng Li <lh3@sanger.ac.uk>

Usage:   bwa <command> [options]

Command: index         index sequences in the FASTA format
         mem           BWA-MEM algorithm
         fastmap       identify super-maximal exact matches
         pemerge       merge overlapping paired ends (EXPERIMENTAL)
         aln           gapped/ungapped alignment
         samse         generate alignment (single ended)
         sampe         generate alignment (paired ended)
         bwasw         BWA-SW for long queries

         shm           manage indices in shared memory
         fa2pac        convert FASTA to PAC format
         pac2bwt       generate BWT from PAC
         pac2bwtgen    alternative algorithm for generating BWT
         bwtupdate     update .bwt to the new format
         bwt2sa        generate SA from BWT and Occ

Note: To use BWA, you need to first index the genome with `bwa index'.
      There are three alignment algorithms in BWA: `mem', `bwasw', and
      `aln/samse/sampe'. If you are not sure which to use, try `bwa mem'
      first. Please `man ./bwa.1' for the manual.

...

Note that bwa writes its (binary) output to standard output by default, so we need to redirect that to a .sai file.

We For simplicity, we will just execute these commands directly (not in a batch job), but since they are fairly large files we will first set up an interactive development (idev) session, which will give us a compute node for 3 hours:, one at a time. Each command should only take few minutes and you will see bwa's progress messages in your terminal.

Code Block

language	bash
title	Start an idev session

idev -p normal -m 180 -N 1 -n 24 -A UT-2015-05-18 --reservation=intro_NGS

Tip
You can tell you're in a idev session because the hostname command will return a compute node name (e.g. nid00438) instead of a login node name (e.g. login5).

For simplicity, we will just execute these commands directly, one at a time. Each command should only take few minutes and you will see bwa's progress messages in your terminal.

bwa aln commands for yeast R1 and R2

# If not already loaded:
module load biocontainers
module load bwa

cd $SCRATCH/core_ngs/alignment/yeast_bwa
bwa aln sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R1.cat.fastq

Code Block

language	bash
title	bwa aln commands for yeast R1 and R2

module load bwa
cd $SCRATCH/core_ngs/alignment/yeast_bwa
bwa aln sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R1.cat.fastq.gz > yeast_R1.sai
bwa aln sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R2.cat.fastq.gz > yeast_R2.sai

When all is done you should have two .sai files: yeast_R1.sai and yeast_R2.sai.

Tip

title	Make sure your output files are not empty

Double check that output was written by doing ls -lh and making sure the file sizes listed are not 0.

Exercise: How long did it take to align the R2 file?

Expand

title	Answer

The last few lines of bwa's execution output should look something like this:

Code Block

language	bash

[bwa_aln_core] 52428817bp sequences have been processed.reads: max_diff = 2
[bwa_aln_core] calculate38bp SA coordinate... 12.86 secreads: max_diff = 3
[bwa_aln_core] write64bp to the disk... 0.00 secreads: max_diff = 4
[bwa_aln_core] 59218093bp sequences have been processed.
[main] Version: 0.7.16a-r1181
[main] CMD: bwa aln sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R2.cat.fastq.gz
[main] Real time: 109.161 sec; CPU: 108.848 sec

So the R2 alignment took ~109 seconds (1.8 minutes).

Since you have your own private compute node, you can use all its resources. It has 24 cores, so re-run the R2 alignment asking for 20 execution threads.

Code Block
bwa aln -t 20 sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R2.cat.fastq.gz > yeast_R2.sai

Exercise: How much of a speedup did you seen when aligning the R2 file with 20 threads?

Expand

title	Answer

The last few lines of bwa's execution output should look something like this:

Code Block

language	bash

reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln_core] calculate SA coordinate... 50.76 sec
[bwa_aln_core] write to the disk... 0.07 sec
[bwa_aln_core] 262144 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 50.35 sec
[bwa_aln_core] write to the disk... 0.07 sec
[bwa_aln_core] 524288 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 1913.5664 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 592180 sequences have been processed.
[main] Version: 0.7.16a17-r1181r1188
[main] CMD: bwa /usr/local/bin/bwa aln -t 20 sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R2R1.cat.fastq.gz
[main] Real time: 9122.655936 sec; CPU: 142123.968597 sec

So the R2 alignment took only ~10 ~123 seconds (real time), or 10+ times as fast as with only one processing thread.

Note, though, that the CPU time with 20 threads was greater (143 sec) than with only 1 thread (109 sec). That's because of the thread management overhead when using multiple threads.

Next we use the bwa sampe command to pair the reads and output SAM format data. Just type that command in with no arguments to see its usage.

For this command you provide the same reference index prefix as for bwa aln, along with the two .sai files and the two original FASTQ files. Also, bwa writes its output to standard output, so redirect that to a .sam file.

Here is the command line statement you need. Just execute it on the command line.

~2 minutes).

Since you have your own private compute node, you can use all its resources. It has 68 cores, so re-run the R2 alignment asking for 60 execution threads.

Code Block

bwa aln -t 60 sacCer3/sacCer3.fa

Code Block

language	bash
title	Pairing of BWA R1 and R2 aligned reads

bwa sampe sacCer3/sacCer3.fa yeast_R1.sai yeast_R2.sai \
  fastq/Sample_Yeast_L005_R1R2.cat.fastq.gz \
  fastq/Sample_Yeast_L005_R2.cat.fastq.gz > yeast_pairedend.sam

You should now have a SAM file (yeast_pairedend.sam) that contains the alignments. It's just a text file, so take a look with head, more, less, tail, or whatever you feel like. Later you'll learn additional ways to analyze the data with samtools once you create a BAM file.

> yeast_R2.sai

Exercise: How much of a speedup did you seen when aligning the R2 file with 20 threadsExercise: What kind of information is in the first lines of the SAM file?

Expand

title	Answer

The SAM file has a number of header lines, which all start with an at sign ( @ ).

The @SQ lines describe each contig (chromosome) and its length.

There is also a @PG line that describes the way the bwa sampe was performed.

Exercise: How many alignment records (not header records) are in the SAM file?

last few lines of bwa's execution output should look something like this:

Expand

title	Hint

This looks for the pattern '^HWI' which is the start of every read name (which starts every alignment record).
Remember -c says just count the records, don't display them.

Code Block

language	bash

grep -P -c '^HWI' yeast_pairedend.sam

Or use the -v (invert) option to tell grep to print all lines that don't match a particular pattern; here, all header lines, which start with @.

Code Block

language	bash

grep -P -v -c '^@' yeast_pairedend.sam

Expand

title	Answer

There are 1,184,360 alignment records.

Exercise: How many sequences were in the R1 and R2 FASTQ files combined?

[bwa_aln] 17bp reads: max_diff = 2
[bwa_aln] 38bp reads: max_diff = 3
[bwa_aln] 64bp reads: max_diff = 4
[bwa_aln] 93bp reads: max_diff = 5
[bwa_aln] 124bp reads: max_diff = 6
[bwa_aln] 157bp reads: max_diff = 7
[bwa_aln] 190bp reads: max_diff = 8
[bwa_aln] 225bp reads: max_diff = 9
[bwa_aln_core] calculate SA coordinate... 266.70 sec
[bwa_aln_core] write to the disk... 0.04 sec
[bwa_aln_core] 262144 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 268.94 sec
[bwa_aln_core] write to the disk... 0.03 sec
[bwa_aln_core] 524288 sequences have been processed.
[bwa_aln_core] calculate SA coordinate... 72.26 sec
[bwa_aln_core] write to the disk... 0.01 sec
[bwa_aln_core] 592180 sequences have been processed.
[main] Version: 0.7.17-r1188
[main] CMD: /usr/local/bin/bwa aln -t 60 sacCer3/sacCer3.fa fastq/Sample_Yeast_L005_R2.cat.fastq.gz
[main] Real time: 19.872 sec; CPU: 617.095 sec

So the R2 alignment took only ~20 seconds (real time), or 6+ times as fast as with only one processing thread.

Note, though, that the CPU time with 60 threads was greater (617 sec) than with only 1 thread (124 sec). That's because of the thread management overhead when using multiple threads.

Next we use the bwa sampe command to pair the reads and output SAM format data. Just type that command in with no arguments to see its usage.

For this command you provide the same reference index prefix as for bwa aln, along with the two .sai files and the two original FASTQ files. Also, bwa writes its output to standard output, so redirect that to a .sam file.

Here is the command line statement you need. Just execute it on the command line.

Code Block

language	bash
title	Pairing of BWA R1 and R2 aligned reads

bwa sampe sacCer3/sacCer3.fa yeast_R1.sai yeast_R2.sai \
  fastq/Sample_Yeast_L005_R1.cat.fastq.gz \
  fastq/Sample_Yeast_L005_R2.cat.fastq.gz > yeast_pairedend.sam

You should now have a SAM file (yeast_pairedend.sam) that contains the alignments. It's just a text file, so take a look with head, more, less, tail, or whatever you feel like. Later you'll learn additional ways to analyze the data with samtools once you create a BAM file.

Exercise: What kind of information is in the first lines of the SAM file?

Expand

title	Answer

The SAM file has a number of header lines, which all start with an at sign ( @ ).

The @SQ lines describe each contig (chromosome) and its length.

There is also a @PG line that describes the way the bwa sampe was performed.

Exercise: How many alignment records (not header records) are in the SAM file?

Expand

title	Hint

This looks for the pattern '^HWI' which is the start of every read name (which starts every alignment record).
Remember -c says just count the records, don't display them.

Code Block

language	bash

grep -P -c '^HWI' yeast_pairedend.sam

Or use the -v (invert) option to tell grep to print all lines that don't match a particular pattern; here, all header lines, which start with @.

Code Block

language	bash

grep -P -v -c '^@' yeast_pairedend.sam

Expand

title	Answer

There are 1,184,360 alignment records.

Exercise: How many sequences were in the R1 and R2 FASTQ files combined?

Expand

title	Hint

zcat fastq/Sample_Yeast_L005_R[12].cat.fastq.gz | wc -l | awk '{print $1/

Expand

title	Hint

zcat fastq/Sample_Yeast_L005_R[12].cat.fastq.gz | wc -l | awk '{print $1/4}'

Expand

title	Answer

There were a total of 1,184,360 original sequences (R1s + R2s)

...

Expand

title	Answer

The expression above returns 612,968. There were 1,184,360 records total, so the percentage is:

Code Block

language	bash
title	Calculate alignment rate

awk 'BEGIN{print 612968/1184360}'

or about 51%. Not great.

Note we perform this calculation in awk's BEGIN block, which is always executed, instead of the body block, which is only executed for lines of input. And here we call awk without piping it any input.

...

The SAMtools program is a commonly used set of tools that allow a user to manipulate SAM/BAM files in many different ways, ranging from simple tasks (like SAM/BAM format conversion) to more complex functions (like sorting, indexing and statistics gathering). It is available in the TACC module system (as well as in BioContainers). Load that module and see what samtools has to offer:functions (like sorting, indexing and statistics gathering). It is available in the TACC module system (as well as in BioContainers). Load that module and see what samtools has to offer:

Expand

title	Make sure you're in a idev session

Make sure you're in an idev session. If you're in an idev session, the hostname command will display a name like c455-021.stampede2.tacc.utexas.edu. But if you're on a login node the hostname will be something like login3.stampede2.tacc.utexas.edu.

If you're on a login node, start an idev session like this:

Code Block

language	bash
title	Start an idev session

idev -p development -m 120 -A UT-2015-05-18 -N 1 -n 68 --reservation=BIO_DATA_week_1

Code Block

language	bash

# If not already loaded
module load biocontainers  # takes a while

module load samtools
samtools

Code Block

title	SAMtools suite usage

Program: samtools (Tools for alignments in the SAM format)
Version: 1.610 (using htslib 1.610)

Usage:   samtools <command> [options]

Commands:
  -- Indexing
     dict           create a sequence dictionary file
     faidx          index/extract FASTA
     fqidx          index/extract FASTQ
     index          index alignment

  -- Editing
     calmd          recalculate MD/NM tags and '=' bases
     fixmate        fix mate information
     reheader       replace BAM header
     rmdup          remove PCR duplicates
     targetcut      cut fosmid regions (for fosmid pool only)
     addreplacerg   adds or replaces RG tags
     markdup        mark duplicates

  -- File operations
     collate        shuffle and group alignments by name
     cat            concatenate BAMs
     merge          merge sorted alignments
     mpileup        multi-way pileup
     sort           sort alignment file
     split          splits a file by read group
     quickcheck     quickly check if SAM/BAM/CRAM file appears intact
     fastq          converts a BAM to a FASTQ
     fasta          converts a BAM to a FASTA

  -- Statistics
     bedcov         read depth per BED region
     coverage       alignment depth and percent coverage
     depth          compute the depth
     flagstat       simple stats
     idxstats       BAM index stats
     phase          phase heterozygotes
     stats          generate stats (former bamcheck)

  -- Viewing
     flags          explain BAM flags
     tview          text alignment viewer
     view           SAM<->BAM<->CRAM conversion
     depad          convert padded BAM to unpadded BAM

...

Warning

title	Know your samtools version!

There are two main "eras" of SAMtools development:

"original" samtools
- v 0.1.19 is the last stable version
"modern" samtools
- v 1.0, 1.1, 1.2 – avoid these (very buggy!)
- v 1.3+ – finally stable!

Unfortunately, some functions with the same name in both version eras have different options and arguments! So be sure you know which version you're using. (The samtools version is usually reported at the top of its usage listing).the top of its usage listing).

TACC BioContainers also offers the original samtools version: samtools/ctrThe default version in the ls5 module system is a "modern" version, but the BioITeam has a copy of the version 0.1.19 samtools for programs that might need it: /work/projects/BioITeam/ls5/bin/samtools-0.1.19. That version is also available as a TACC BioContainers module.--3.

samtools view

The samtools view utility provides a way of converting between SAM (text) and BAM (binary, compressed) format. It also provides many, many other functions which we will discuss lster. To get a preview, execute samtools view without any other arguments. You should see:

Code Block

title	samtools view usage

Usage: samtools view [options] <in.bam>|<in.sam>|<in.cram> [region ...]

Options:
  -b       output BAM
  -C       output CRAM (requires -T)
  -1       use fast BAM compression (implies -b)
  -u       uncompressed BAM output (implies -b)
  -h       include header in SAM output
  -H       print SAM header only (no alignments)
  -c       print only the count of matching records
  -o FILE  output file name [stdout]
  -U FILE  output reads not selected by filters to FILE [null]
  -t FILE  FILE listing reference names and lengths (see long help) [null]
  -X       include customized index file
  -L FILE  only include reads overlapping this BED this BED FILE [null]
  -r STR   only include reads in read group STR [null]
  -R FILE  only include reads with read group listed in FILE [null]
  -rd STR:STR
           only include reads in read group with tag STR and associated value STR [null]
  -RD STR:FILE
           only include reads with read grouptag STR and associated values listed in
           FILE [null]
  -q INT   only include reads with mapping quality >= INT [0]
  -l STR   only include reads in library STR [null]
  -m INT   only include reads with number of CIGAR operations consuming
           query sequence >= INT [0]
  -f INT   only include reads with all  of the FLAGs in INT present [0]
  -F INT   only include reads with none of the FLAGS in INT present [0]
  -G INT   only EXCLUDE reads with all  of the FLAGs in INT present [0]
  -s FLOAT subsample reads (given INT.FRAC option value, 0.FRAC is the
 the
           fraction of templates/read pairs to keep; INT part sets seed)
  -M       use the multi-region iterator (increases the speed, removes
           duplicates and fractionoutputs ofthe templates/readreads pairsas tothey keep;are INTordered partin setsthe seedfile)
  -x STR   read tag to strip (repeatable) [null]
  -B       collapse the backward CIGAR operation
  -?       print long help, including note about region specification
  -S       ignored (input format is auto-detected)
  --no-PG  do not add a PG line
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
  -T, --reference FILE
               Reference sequence FASTA FILE [null]
  -@, --threads INT
 FILE [null]
  -@, --threads INT
               Number of additional threads to use [0]
      --write-index
               Automatically index the output files [off]
      --verbosity INT
               Set Numberlevel of additional threads to use [0]verbosity

That is a lot to process! For now, we just want to read in a SAM file and output a BAM file. The input format is auto-detected, so we don't need to specify it (although you do in v0.1.19). We just need to tell the tool to output the file in BAM format, and to include the header records.

...

Code Block

title	samtools sort usage

Usage: samtools sort [options...] [in.bam]
Options:
  -l INT     Set compression level, from 0 (uncompressed) to 9 (best)
  -m INT     Set maximum memory per thread; suffix K/M/G recognized [768M]
  -n         Sort by read name
  -t TAG     Sort by value of TAG. Uses position as secondary index (or read name if -n is set)
  -o FILE    Write final output to FILE rather than standard output
  -T PREFIX  Write temporary files to PREFIX.nnnn.bam
  --no-PG    do not add a PG line
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]
  -@, --threads INT
               Number of additional threads to use [0]
      --verbosity INT
               Set level of verbosity

In most cases you will be sorting a BAM file from name order to locus order. You can use either -o or redirection with > to control the output.

...

Now that you've done everything the hard way, let's see how to do run an alignment pipeline using a BWA alignment script maintained by the BioITeam, /workwork2/projects/BioITeam/common/script/align_bwa_illumina.sh. Type in the script name to see its usage.

...

We're going to run this script and a similar Bowtie2 alignment script, on the yeast data using the TACC batch system. In a new directory, copy over the commands and submit the batch job. We ask for 1 hour 2 hours (-t 0102:00:00) with 4 tasks/node (-w 4); since we have 4 commands, this will run on 1 compute node.

...

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 startslooks withlike "nidc455-004.stampede2.tacc.utexas.edu", exit the idev session

# 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 -s -f ../fastq

# Copy the alignment commands file and submit the batch job
cp $CORENGS/tacc/aln_script.cmds .
launcher_creator.py -j aln_script.cmds -n aln_script -t 0102:00:00 -w 4 -a UT-2015-05-18 -q normal
sbatch --reservation=intro_NGSBIO_DATA_week_1 aln_script.slurm 
showq -u

...

Code Block

language	bash
title	Commands to run multiple alignment scripts

/workwork2/projects/BioITeam/common/script/align_bwa_illumina.sh     global ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bwa_global sacCer3 1 50
/workwork2/projects/BioITeam/common/script/align_bwa_illumina.sh     local  ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bwa_local  sacCer3 1
/workwork2/projects/BioITeam/common/script/align_bowtie2_illumina.sh global ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bt2_global sacCer3 1 50
/workwork2/projects/BioITeam/common/script/align_bowtie2_illumina.sh local  ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bt2_local  sacCer3 1

...

The great thing about pipeline scripts like this is that you can perform alignments on many datasets in parallel at TACC, and they are written to take advantage of having multiple cores on TACC nodes where possible.

On the Lonestar5 stampede2, with its 24 68 physical cores per node, they are designed to run best with no more than 4 tasks per node.

Tip

title	Always specify wayness 4 for alignment pipeline scripts

These alignment scripts should always be run with a wayness of 4 (-w 4) in the Lonestar5 stampede2 batch system, meaning at most 4 commands per node.

...

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Key

Stage the alignment data

samtools view