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Exercise #3: PE alignment with BioITeam scripts

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,  /work2work/projects/BioITeam/common/script/align_bwa_illumina.sh. Type in the script name to see its usage.

align_bwa_illumina.sh 20212022_0605_05
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

...

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 2 hours (-t 02:00:00) with 4 tasks/node (-w 4); since we have 4 commands, this will run on 1 compute node.

# Make sure you're not in an idev session by looking at the hostname
hostname
# If the hostname looks like "c455-004.stampede2ls6.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 -s -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 .

launcher_creator.py -j aln_script.cmds -n aln_script -t 0201:00:00 -w 4 -a UT-2015-05-18OTH21164 -q normal
sbatch --reservation=BIO_DATA_week_1CoreNGS-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

While we're waiting for the job to complete, lets look at the aln_script.cmds file.

/work2work/projects/BioITeam/common/script/align_bwa_illumina.sh     global ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bwa_global sacCer3 1 50
/work2work/projects/BioITeam/common/script/align_bwa_illumina.sh     local  ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bwa_local  sacCer3 1
/work2work/projects/BioITeam/common/script/align_bowtie2_illumina.sh global ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bt2_global sacCer3 1 50
/work2work/projects/BioITeam/common/script/align_bowtie2_illumina.sh local  ./fastq/Sample_Yeast_L005_R1.cat.fastq.gz bt2_local  sacCer3 1

...

• Hard trims FASTQ, if optionally specified (fastx_trimmer)
  
• Performs the global or local alignment (here, a PE alignment)
  
  • BWA global: bwa aln the R1 and R2 separately, then bwa sampe to produce a SAM file
    
  • BWA local: call bwa mem with both R1 and R2 to produce a SAM file
    
  • Bowtie2 global: call bowtie2 in its default global (end--global with to-end) mode on both R1 and R2 to produce a SAM file
    
  • Bowtie2 local: call bowtie2 --local with both R1 and R2 to produce a SAM file
• Converts SAM to BAM (samtools view)
• Sorts the BAM (samtools sort)
• Marks duplicates (Picard MarkDuplicates)
• Indexes the sorted, duplicate-marked BAM (samtools index)
• Gathers statistics (samtools idxstats, samtools flagstat, plus a custom statistics script of Anna's)
• Removes intermediate files
  

...

This will show something like:

..Done ------------------------------------------------------------------
..Done alignmentUtils.pl bamstats - 20202022-06-1410 2312:1959:3805
.. samstats file 'bwa_global.samstats.txt' exists and is not empty - 20202022-06-1410 2312:1959:3805
===============================================================================
## Cleaning up files (keep 0) - 20202022-06-1410 2312:1959:3805
===============================================================================
ckRes 0 cleanup
===============================================================================
## All bwa alignment tasks completed successfully! - 20202022-06-1410 2312:1959:3806
===============================================================================

...

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 stampede2, with its 68 physical cores per node, they the ls6 the pipeline scripts are designed to run best with no more than 4 tasks per node. Although each ls6 node has 128 physical cores per node, the alignment workflow is heavily I/O bound overall, and we don't want to overload the file system.

Exercise #4: Bowtie2

...

alignment - Vibrio cholerae RNA-seq

While we have focused on aligning eukaryotic data, the same tools can be used with prokaryotic data. The major differences are less about the underlying data and much more about the external/public databases that store and distribute reference data. If we want to study a prokaryote, the reference data is usually downloaded from a resource like GenBank.  

...

1. Prepare the vibCho reference index for bowtie2 from GenBank records
2. Align reads using bowtie2, producing a SAM file
3. Convert the SAM file to a BAM file (samtools view) 
4. Sort the BAM file by genomic location (samtools sort)
5. Index the BAM file (samtools index)
6. Gather simple alignment statistics (samtools flagstat and samtools idxstatidxstats)

Obtaining the GenBank records

First prepare a directory to work infor the vibCho fasta, and change to it:

mkdir -p $SCRATCH/core_ngs/references/vibChofasta
cd $SCRATCH/core_ngs/references/vibChofasta

V. cholerae has two chromosomes. We download each separately.

1. Navigate to http://www.ncbi.nlm.nih.gov/nuccore/NC_012582
   • click on the Send to down arrow (top right of page)
     • select Complete Record
     • select File as Destination, and Format FASTA
     • click Create File
       
   • in the Opening File dialog, select Save File then OK
     • Save the file on your local computer as NC_012582.fa
2. Back on the main http://www.ncbi.nlm.nih.gov/nuccore/NC_012582 page
   • click on the Send to down arrow (top right of page)
     • select Complete Record
     • select File as Destination, and Format GFF3
     • click Create File
       
   • in the Opening File dialog, select Save File then OK
     • Save the file on your local computer as NC_012582.gff3
3. Repeat steps 1 and 2 for the 2nd chromosome
   
   • NCBI URL is http://www.ncbi.nlm.nih.gov/nuccore/NC_012583
   • use NC_012583 as the filename prefix for the files you save
   • you should now have 4 files:
     • NC_012582.fa, NC_012582.gff3
     • NC_012583.fa, NC_012583.gff3
       
4. Transfer the files from your local computer to TACC
   • to the ~/scratch/core_ngs/references/vibCho directory created above
     
     • On a Mac or Windows with a WSL shell10 or later, use scp from your laptop
       
     • On Otherwise on Windows, use the pscp.exe PuTTy tool
     • See Copying files between TACC and your laptop

...

mkdir -p $SCRATCH/core_ngs/references/vibChofasta
cd $SCRATCH/core_ngs/references/vibChofasta
cp $CORENGS/ncbireferences/vibChofasta/NC_* .

Once you have the 4 files locally in your $SCRATCH/core_ngs/references/vibCho directory, combine them using cat:

cd $SCRATCH/core_ngs/references/vibChofasta
cat NC_01258[23].fa   > vibCho.O395.fa
cat NC_01258[23].gff3 > vibCho.O395.gff3

# verify there are 2 contigs in vibCho.O395.fa
grep -P '^>' vibCho.O395.fa

Now we have a reference sequence file that we can use with the bowtie2 reference builder, and ultimately align sequence data against.

...

idev -m 180120 -A OTH21164 -pN normal1 -Ar UT-2015-05-18CoreNGS-Thu
# or
idev -m 90 -A OTH21164 -N 1 -np 68development 

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

module load biocontainers
module load bowtiebowtie2

 Now that it's loaded, check out the options. There are a lot of them! In fact for the full range of options and their meaning, Google "Bowtie2 manual" and bring up that page (http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml). The Table of Contents is several pages long! Ouch!

...

First create a directory specifically for the bowtie2 index, then build the index using bowtie-build.

mkdir -p $SCRATCH/core_ngs/references/fasta
cd $SCRATCH/core_ngs/references/fasta
cp $CORENGS/references/fasta/vibCho* .

mkdir -p $SCRATCH/core_ngs/references/bt2/vibCho
cd $SCRATCH/core_ngs/references/bt2/vibCho

# Symlink to the fasta file you created using relative path syntax
ln -sf $SCRATCH/core_ngs/references../../fasta/vibCho.O395.fa
# or, to catch up:
ln -sf $CORENGS/references/vibCho
bowtie2-build vibCho.O395.fa

bowtie2-build vibCho.O395.fa vibCho.O395

This should also go pretty fast. You can see the resulting files using ls like before.

Performing the bowtie2 alignment

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

#idev Get-m the120 FASTQ-A toOTH21164 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/vibChoN 1 -r CoreNGS-Thu
# or
idev -m 90 -A OTH21164 -N 1 -p development

module load biocontainers
module load bowtie2

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

idev# -pGet normala pre-mbuilt 120vibCho -A UT-2015-05-18 -N 1 -n 68
module load biocontainers
module load bowtieindex if you didn't already build one
mkdir -p $SCRATCH/core_ngs/references/bt2/vibCho
cp $CORENGS/references/bt2/vibCho/*.*  $SCRATCH/core_ngs/references/bt2/vibCho/

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

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

...

We'll be aligning the V. cholerae reads now in ./fq/cholera_rnaseq.fastq.gz (how many sequences does it contain?)

...

So execute this bowtie2 global, single-end alignment command:

mkdir -p $SCRATCH/core_ngs

Notes:

/references/fasta
cp $CORENGS/references/fasta/vibCho*  $SCRATCH/core_ngs/references/fasta/

mkdir -p $SCRATCH/core_ngs/references/bt2/vibCho
cp $CORENGS/references/bt2/vibCho/*.*  $SCRATCH/core_ngs/references/bt2/vibCho/

mkdir -p $SCRATCH/core_ngs/alignment/vibCho
cd $SCRATCH/core_ngs/alignment/vibCho
ln -sf ../../references/bt2/vibCho
ln -sf ../../alignment/fastq fqmkdir -p $SCRATCH/core_ngs/alignment/vibCho

cd $SCRATCH/core_ngs/alignment/vibCho
bowtie2 -x vibCho/vibCho.O395 -U fq/cholera_rnaseq.fastq.gz \
  -S cholera_rnaseq.sam 2>&1 | tee aln_global.log

Notes:

• -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
• -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)

Since the FASTQ file is not large, this should not take too long, and you will see progress output like this:

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.

...

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

After bowtie2 came out with a local alignment option, it wasn't long before bwa developed its own local alignment algorithm called BWA-MEM (for Maximal Exact Matches), implemented by the bwa mem command. 

bwa mem has the following advantages:

• It provides the simplicity of using bwa without the complexities of local alignment
• It can align different portions of a read to different locations on the genome
  • In a total RNA-seq experiment, reads will (at some frequency) span a splice junction themselves
    • or a pair of reads in a paired-end library will fall on either side of a splice junction.
  • We want to be able to align these splice-adjacent reads for many reasons, from accurate transcript quantification to novel fusion transcript discovery.

This exercise will align a human total RNA-seq dataset that includes numerous reads that cross splice junctions.

A word about real splice-aware aligners

Using bwa mem for RNA-seq alignment is sort of a "poor man's" RNA-seq alignment method. Real splice-aware aligners like tophat2, hisat2 or STAR have more complex algorithms (as shown below) – and take a lot more time!

Image Added

In the transcriptome-aware alignment above, reads that span splice junctions are reported in the SAM file with genomic coordinates that start in the first exon and end in the second exon (the CIGAR string uses the N operator, e.g. 30M1000N60M).

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 of the two will be marked as secondary (0x100 flag).

Setup for BWA mem

First set up our working directory for this alignment. Since it takes a long time to build a bwa index for a large genome (here human hg38/GRCh38), we'll use one that the BioITeam maintains in its /work/projects/BioITeam/ref_genome area.

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

# 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). The most important parameters are the following:

RNA-seq alignment with bwa mem

Based on its help info, this is the structure of the bwa mem command we will use:

bwa mem -M <ref.fa> <reads.fq> > outfile.sam

After performing the setup above, execute the following command in your idev session:

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

This multi-pipe command performs three steps:

• The bwa mem alignment
  • the program's progress output (on standard error) is redirected to a log file (2>hs_rna.bwamem.log)
  • its alignment records (on standard output) is piped to the next step (conversion to BAM)
• Conversion of bwa mem's SAM output to BAM format
  • recall that the -b option to samtools view says to output in BAM format
• Sorting the BAM file
  • samtools sort takes the binary output from samtools view and writes a sorted BAM file.

Because the progress output is being redirected to a log file, we won't see anything until the command completes. Then you should have a human_rnaseq.sort.bam file and an hs_rna.bwamem.log logfile.

Exercise: Compare the number of original FASTQ reads to the number of alignment records.

cd $SCRATCH/core_ngs/alignment/bwamem
zcat ./fastq/human_rnaseq.fastq.gz | wc -l | awk '{print $1/4}'

cd $SCRATCH/core_ngs/alignment/bwamem
samtools flagstat human_rnaseq.sort.bam | tee hs_rnaseq.flagstat.txt

133570 + 0 in total (QC-passed reads + QC-failed reads)
33570 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
133450 + 0 mapped (99.91% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)