Hello,
I have a problem with samtools newest version and BWA tool from Sourceforge.
I have sam files, but it looks like samtools are not recognizing header,
and also not doing indexing. Is that a problem with bwa alignment, or
samtools? scripts and samtools error message example are attached.
--
Best regards
Baiba Alkšere
#!/bin/bash -x
module load bio/samtools/1.10
export INPATH=/dir
export INPATH1=/dir
export INPATH2=/dir
export INPATH3=/dir
export INPATH4=/dir
export INPATH5=/dir
export INPATH6=/dir
export INPATH7=/dir
export reference=/_refseq.fasta
samtools faidx $reference
for file in $INPATH/*.sam ;
do
bname=$(basename $file ".sam")
echo $bname
file=$INPATH/*.sam
echo file: $file
bamfile=$INPATH1/$bname".bam"
namesort=$INPATH2/$bname"_sorted.bam"
fixmate=$INPATH3/$bname"_sorted_fixmate.bam"
positionsort=$INPATH4/$bname"_sorted_fixmate_position.bam"
markdup=$INPATH5/$bname"_sorted_fixmate_position_markdup.bam"
bai=$INPATH6/$bname".bai"
stats=$INPATH7/$bname".txt"
echo $bamfile $namesort $fixmate $positionsort $markdup
samtools view -bT $reference $file > $bamfile
samtools sort -@ 12 -n $bamfile > $namesort
samtools fixmate -m $namesort > $fixmate
samtools sort -@ 12 $fixmate > $positionsort
samtools markdup $positionsort > $markdup
samtools index > $markdup
samtools flagstat $markdup > $stats
done
+ samtools sort -@ 12 -n file.bam
samtools sort: failed to read header from "file.bam"
+ samtools fixmate -m file_sorted.bam
Usage: samtools fixmate <in.nameSrt.bam> <out.nameSrt.bam>
Options:
-r Remove unmapped reads and secondary alignments
-p Disable FR proper pair check
-c Add template cigar ct tag
-m Add mate score tag
--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
As elsewhere in samtools, use '-' as the filename for stdin/stdout. The input
file must be grouped by read name (e.g. sorted by name). Coordinated sorted
input is not accepted.
+ samtools sort -@ 12 file_sorted_fixmate.bam
samtools sort: failed to read header from "file_sorted_fixmate.bam"
+ samtools markdup file_sorted_fixmate_position.bam
Usage: samtools markdup <input.bam> <output.bam>
Option:
-r Remove duplicate reads
-l INT Max read length (default 300 bases)
-S Mark supplementary alignments of duplicates as duplicates
(slower).
-s Report stats.
-f NAME Write stats to named file. Implies -s.
-T PREFIX Write temporary files to PREFIX.samtools.nnnn.nnnn.tmp.
-d INT Optical distance (if set, marks with dt tag)
-c Clear previous duplicate settings and tags.
-m --mode TYPE Duplicate decision method for paired reads.
TYPE = t measure positions based on template start/end
(default).
s measure positions based on sequence start.
--include-fails Include quality check failed reads.
--no-PG Do not add a PG line
-t Mark primary duplicates with the name of the original in a
'do' tag. Mainly for information and debugging.
--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]
--write-index
Automatically index the output files [off]
--verbosity INT
Set level of verbosity
The input file must be coordinate sorted and must have gone through fixmates
with the mate scoring option on.
+ samtools index
+ samtools flagstat file_sorted_fixmate_position_markdup.bam
Failed to read header for "file_sorted_fixmate_position_markdup.bam"
#!/bin/bash -x
INPATH=dir
OUTPATH=dir
reference=refseq.fasta
cd //tools/bwa-0.7.17
./bwa index $reference
for file1 in $INPATH/*.fastq.gz ;
do
bname=$(basename $file1 )
sample=$( cut -d'_' -f 1,2,3<<<"$bname")
echo $bname $sample
input1=$INPATH/$sample"_R1_001.paired.fastq.gz"
output1=$OUTPATH/$sample"_R1_001.paired.sai"
input2=$INPATH/$sample"_R2_001.paired.fastq.gz"
output2=$OUTPATH/$sample"_R2_001.paired.sai"
outfile=$OUTPATH/$sample".sam"
echo $input1 $output1 $input2 $output2
./bwa mem -t 8 $reference $input1 $input2 | gzip -3 > $outfile
echo $outfile
done
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