@RG information from Bowtie2 Alignments

Question

[landrjos]

Hi All,

I use Bowtie2 to align my Illumina Fastq sequence files then convert to BAM using Samtools. The BAM output file does not have any barcode information. Or any @RG information at all. Here is the header from one of the BAM files....

Does Bowtie2 save this information? If so I would I access it from the BAM file?

@HD VN:1.0  SO:coordinate
@SQ SN:chr1 LN:197195432
@SQ SN:chr2 LN:181748087
@SQ SN:chr3 LN:159599783
@SQ SN:chr4 LN:155630120
@SQ SN:chr5 LN:152537259
@SQ SN:chr6 LN:149517037
@SQ SN:chr7 LN:152524553
@SQ SN:chr8 LN:131738871
@SQ SN:chr9 LN:124076172
@SQ SN:chr10    LN:129993255
@SQ SN:chr11    LN:121843856
@SQ SN:chr12    LN:121257530
@SQ SN:chr13    LN:120284312
@SQ SN:chr14    LN:125194864
@SQ SN:chr15    LN:103494974
@SQ SN:chr16    LN:98319150
@SQ SN:chr17    LN:95272651
@SQ SN:chr18    LN:90772031
@SQ SN:chr19    LN:61342430
@SQ SN:chrX LN:166650296
@SQ SN:chrY LN:15902555
@SQ SN:chrM LN:16299
@SQ SN:chr13_random LN:404305
@SQ SN:chr17_random LN:628739
@SQ SN:chr1_random  LN:1231697
@SQ SN:chr3_random  LN:41899
@SQ SN:chr4_random  LN:160594
@SQ SN:chr5_random  LN:357350
@SQ SN:chr7_random  LN:362490
@SQ SN:chr8_random  LN:849593
@SQ SN:chr9_random  LN:449403
@SQ SN:chrUn_random LN:5900358
@SQ SN:chrX_random  LN:1785075
@SQ SN:chrY_random  LN:58682461
@PG ID:bowtie2  PN:bowtie2  VN:2.1.0
HWI-ST425:160:D1JFWACXX:3:1306:14534:76146  16  chr1    3000195 42  36M *   0   0   GTATTATAATTGTAATAGTATATACTTGTATGTACT    JJJJJJJJJJJIJJJJJJJJJJHFHHHHFFFFFBCC    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:MD:Z:36    YT:Z:UU
HWI-ST425:221:C6B83ACXX:8:1201:13332:28408  0   chr1    3000206 42  36M *   0   0   GTAATAGTATATACTTGTATGTACTTAAAATATTTT    CBCFFFFDHHHHHJJJJIJJJJJJJJJJJJJJJJJJ    AS:i:-4 XN:i:4  XM:i:4  XO:i:0  XG:i:0  NM:i:MD:Z:32N0N0N0N0    YT:Z:UU
HWI-ST425:221:C6B83ACXX:8:1113:12482:87912  0   chr1    3000818 42  36M *   0   0   CTATCATGACCTCTGAATGACTAGGGAATCTTGGAC    @@@FFDFFHHHHHJJIGIJCHGHHGGIEHHGGIIIJ    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:MD:Z:36    YT:Z:UU
HWI-ST425:221:C6B83ACXX:8:2309:10001:51423  0   chr1    3000818 42  36M *   0   0   CTATCATGACCTCTGAATGACTAGGGAATCTTGGAC    CCCFFFFFHHHHHJJJJJJIIJJJJJJJIJJJJGIJ    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:MD:Z:36    YT:Z:UU

[Devon Ryan]

Did you provide the read group information to bowtie2? If so, show the parameters you used.

[landrjos]

Here is the script I am using to run Bowtie2....Where would I add the read group information?

#!/bin/bash
#shell
#$ -S /bin/bash
#$ -pe smp_2 10

##### export OMPI_MCA_orte_rsh_agent="rsh:ssh"
##### export LD_LIBRARY_PATH=/usr/global/openmpi-1.5.3-w-psm/lib:$LD_LIBRARY_PATH
#####
#
#    variables that need to be set/changed by user
#
#####

######
#
#   unpaired reads:  script is set up to handle up to 5 (use UNPAIRED_READ_1...UNPAIRED_READ_5) to specify basenames 
#
######

export UNPAIRED_READ_1="GEO_Rep2_FAIRE_P1B9_S2_R2_001_Trimmomatic"            ## JOB_NAME WILL be based on read_1
          ## BASENAME for read_2 (no filetype extension)
          ## BASENAME for first file containing unpaired reads

export REFERENCE_GENOME="mm9.RM"    ## genome against which alignments are to be made

#
#   Build unpaired read list from basenames specified above
#   also move various files to $TMPDIR and uncompress if applicable 
#

export UNPAIRED_READS=${TMPDIR}/${UNPAIRED_READ_1}.fastq          ### UNPAIRED_READ_1
gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_1}.fastq
cp ${SGE_O_WORKDIR}/${UNPAIRED_READ_1}.fastq.gz ${TMPDIR}
gunzip ${TMPDIR}/${UNPAIRED_READ_1}.fastq.gz

if env | grep -q ^UNPAIRED_READ_2=
then
  export UNPAIRED_READS=${UNPAIRED_READS},${TMPDIR}/${UNPAIRED_READ_2}.fastq    ### UNPAIRED_READ_2
  gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_2}.fastq
  cp ${SGE_O_WORKDIR}/${UNPAIRED_READ_2}.fastq.gz ${TMPDIR}
  gunzip ${TMPDIR}/${UNPAIRED_READ_2}.fastq.gz
fi

if env | grep -q ^UNPAIRED_READ_3=
then
  export UNPAIRED_READS=${UNPAIRED_READS},${TMPDIR}/${UNPAIRED_READ_3}.fastq    ### UNPAIRED_READ_3
  gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_3}.fastq
  cp ${SGE_O_WORKDIR}/${UNPAIRED_READ_3}.fastq.gz ${TMPDIR}
  gunzip ${TMPDIR}/${UNPAIRED_READ_3}.fastq.gz
fi

if env | grep -q ^UNPAIRED_READ_4=
then
  export UNPAIRED_READS=${UNPAIRED_READS},${TMPDIR}/${UNPAIRED_READ_4}.fastq   ### UNPAIRED_READ_4
  gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_4}.fastq
  cp ${SGE_O_WORKDIR}/${UNPAIRED_READ_4}.fastq.gz ${TMPDIR}
  gunzip ${TMPDIR}/${UNPAIRED_READ_4}.fastq.gz
fi

if env | grep -q ^UNPAIRED_READ_5=
then
  export UNPAIRED_READS=${UNPAIRED_READS},${TMPDIR}/${UNPAIRED_READ_5}.fastq    ### UNPAIRED_READ_5
  gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_5}.fastq
  cp ${SGE_O_WORKDIR}/${UNPAIRED_READ_5}.fastq.gz ${TMPDIR}
  gunzip ${TMPDIR}/${UNPAIRED_READ_5}.fastq.gz
fi
printenv UNPAIRED_READS

### ls -l $TMPDIR
##### export UNPAIRED_READS=${TMPDIR}/${UNPAIRED_READ_1}.fastq,${TMPDIR}/${UNPAIRED_READ_2}.fastq,${TMPDIR}/${UNPAIRED_READ_3}.fastq

#
#   Set up our executable path so that we have mpirun and friends as well as mpiblast and associates in
#      our executable path
#

export BOWTIE2_PATH=/usr/global/bowtie2-2.1.0/bin   ## PATH to bowtie2 suite
export TWOBIT_PATH=/home/jscarsda/bin/x86_64
export PATH=$BOWTIE2_PATH:$PATH

#
#   environment variable BOWTIE2_INDEXES specifies directory where reference genomes reside if not in current directory
#

export BOWTIE2_INDEXES=/home/jlandry/reference_genomes_mm9_masked

#
#    compress the data to be aligned to enable more efficient copying of data to $TMPDIR, which is a unique directory on
#    the /tmp filesystem on the node on which this job is running.  This directory is created by the queueing system for
#    our job.
#

#### gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_1}.fastq
#### gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_2}.fastq
#### gzip ${SGE_O_WORKDIR}/${UNPAIRED_READ_3}.fastq

#
#  copy files containing reads to be aligned to ${TMPDIR} since local I/O is much faster than nfs I/O
#
#

#
#   run bowtie2 to perform alignments
#

time bowtie2-align -p $NSLOTS -N 1 -x $REFERENCE_GENOME -U $UNPAIRED_READS \
                                                        -S ${TMPDIR}/${UNPAIRED_READ_1}.${JOB_ID}.sam --no-unal

#
#    gzip output sam file
#

gzip ${TMPDIR}/${UNPAIRED_READ_1}.${JOB_ID}.sam

#
#   copy compressed sam file back to home directory
#

cp ${TMPDIR}/${UNPAIRED_READ_1}.${JOB_ID}.sam.gz ${SGE_O_WORKDIR}

[Devon Ryan]

Right, so you didn't tell it to use any read group information.

[landrjos]

I am not a professional. Could you suggest some changes to the script to retain the read group information?

[Devon Ryan]

Have you read the bowtie2 documentation? Do so first, this is documented there.