Error using bamCoverage to generate bw file
1
0
Entering edit mode
3 months ago
Zebk • 0

Dear All,

I am trying to generate bw files using bamCoverage. But, everytime it causes errors like this:

bamFilesList: ['./alignment/bam/M-EK-0D-IgG-1_S31_L003.mapped.sorted.bam']
binLength: 50
numberOfSamples: None
blackListFileName: None
skipZeroOverZero: False
bed_and_bin: False
genomeChunkSize: None
defaultFragmentLength: read length
numberOfProcessors: 1
verbose: False
region: None
bedFile: None
minMappingQuality: None
ignoreDuplicates: False
chrsToSkip: []
stepSize: 50
center_read: False
samFlag_include: None
samFlag_exclude: None
minFragmentLength: 0
maxFragmentLength: 0
zerosToNans: False
smoothLength: None
save_data: False
out_file_for_raw_data: None
maxPairedFragmentLength: 1000
Traceback (most recent call last):
  File "/home/anaconda3/bin/bamCoverage", line 12, in <module>
    main(args)
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/bamCoverage.py", line 256, in main
    wr.run(writeBedGraph.scaleCoverage, func_args, args.outFileName,
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/writeBedGraph.py", line 171, in run
    bedGraphToBigWig(chrom_names_and_size, [x[3] for x in res], out_file_name)
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/writeBedGraph.py", line 293, in bedGraphToBigWig
    bw.addHeader(chromSizes, maxZooms=10)
RuntimeError: Received an error while writing the bigWig header
[bwClose] There was an error while finishing writing a bigWig file! The output is likely truncated.

I used the following commands to generate bam files:

# bowtie2 alignment
bowtie2 -p6 --very-sensitive-local --no-mixed --no-discordant --phred33 -I 10 -X 2000 -x ./bt2/chr \
-1 ./trim/${SAMPLE}_R1_001.trimmed.fastq.gz \
-2 ./trim/${SAMPLE}_R2_001.trimmed.fastq.gz \
-S ./alignment/sam/${SAMPLE}_bowtie2.sam &> ./alignment/sam/${SAMPLE}_bowtie2.txt;

# remove duplicate with picard
java -jar picard.jar MarkDuplicates \
I=./alignment/removeDuplicate/${SAMPLE}_bowtie2.sorted.sam \
O=./alignment/removeDuplicate/${SAMPLE}_bowtie2.sorted.rmDup.sam \
REMOVE_DUPLICATES=true \
M=./alignment/removeDuplicate/picard_summary/${SAMPLE}_picard.rmDup.txt;

# Filtering mapped reads by the mapping quality filtering
minQualityScore=2
samtools view -q $minQualityScore ./alignment/removeDuplicate/${SAMPLE}_bowtie2.sorted.rmDup.sam >./alignment/sam/${SAMPLE}_bowtie2.qualityScore2.sam; 

#convert to bam files
samtools view -bS -h -F 0x04 ./alignment/removeDuplicate/${SAMPLE}_bowtie2.sorted.rmDup.sam > ./alignment/bam/${SAMPLE}_bowtie2.mapped.bam; \
samtools sort ./alignment/bam/${SAMPLE}_bowtie2.mapped.bam \
-o ./alignment/bam/${SAMPLE}.mapped.sorted.bam; \
samtools index ./alignment/bam/${SAMPLE}.mapped.sorted.bam \
./alignment/bam/${SAMPLE}.mapped.sorted.bai;

#bamCoverage command
bamCoverage -b ./alignment/bam/${SAMPLE}.mapped.sorted.bam -o ./alignment/bigwig/${SAMPLE}.bw

It sounds like the header problem, but not clear how to resolve it... "RuntimeError: Received an error while writing the bigWig header"

I sincerely appreciate reading this long email and your kind help.

Best,

bw bamCoverage error • 386 views
ADD COMMENT
0
Entering edit mode
3 months ago
ATpoint 60k
samtools view -q $minQualityScore ./alignment/removeDuplicate/${SAMPLE}_bowtie2.sorted.rmDup.sam >./alignment/sam/${SAMPLE}_bowtie2.qualityScore2.sam; 

Add option -h to preserve the header. Or better work with BAM files right away, SAMs have no value tbh.

ADD COMMENT
0
Entering edit mode

Thank you so much. I did not use -h with -q. I will also work with BAM rather than SAM! Thank you again. Best,

ADD REPLY
0
Entering edit mode

Hi ATpoint,

I changed my script like this:

# bowtie2 alignment
bowtie2 -p6 --very-sensitive-local --no-mixed --no-discordant --phred33 -I 10 -X 2000 -x ./bt2/chr \
-1 ./trim/${SAMPLE}_R1_001.trimmed.fastq.gz \
-2 ./trim/${SAMPLE}_R2_001.trimmed.fastq.gz \
-S ./alignment/sam/${SAMPLE}_bowtie2.sam &> ./alignment/sam/${SAMPLE}_bowtie2.txt;

#Filtering mapped reads by the mapping quality filtering
minQualityScore=2

#Filter and keep the mapped read pairs
for SAMPLE in $SAMPLES; \
do samtools view -b -h -q $minQualityScore -F 0x04 ./alignment/sam/${SAMPLE}_bowtie2.sam | samtools sort -o ./alignment/bam/${SAMPLE}.sorted.bam; \
samtools index ./alignment/bam/${SAMPLE}.sorted.bam ./alignment/bam/${SAMPLE}.sorted.bai; \
done

#Remove duplicates 
for SAMPLE in $SAMPLES; \
do java -jar picard.jar MarkDuplicates \
I=./alignment/bam/${SAMPLE}.sorted.bam \
O=./alignment/bam/${SAMPLE}.sorted.rmDup.bam \
REMOVE_DUPLICATES=true \
M=./alignment/bam/picard_summary/${SAMPLE}_picard.rmDup.txt; \
samtools index ./alignment/bam/${SAMPLE}.sorted.rmDup.bam ./alignment/bam/${SAMPLE}.sorted.rmDup.bai; \
#done

#bw file generation
for SAMPLE in $SAMPLES; \
do bamCoverage -b ./alignment/bam/${SAMPLE}.sorted.bam -o ./alignment/bigwig/${SAMPLE}_ori.bw --normalizeUsing RPKM --binSize 10 -p 10 2> ./alignment/bigwig/${SAMPLE}_ori.bw.log;
done

As -h was used with samtools, I expected bamCoverage might go forward but it caused the same error...

normalization: RPKM
bamFilesList: ['./alignment/bam/F-EK-0D-H3K4me3-1_S26_L003.sorted.rmDup.bam']
binLength: 10
numberOfSamples: None
blackListFileName: None
skipZeroOverZero: False
bed_and_bin: False
genomeChunkSize: None
defaultFragmentLength: read length
numberOfProcessors: 6
verbose: False
region: None
bedFile: None
minMappingQuality: None
ignoreDuplicates: False
chrsToSkip: []
stepSize: 10
center_read: False
samFlag_include: None
samFlag_exclude: None
minFragmentLength: 0
maxFragmentLength: 0
zerosToNans: False
smoothLength: None
save_data: False
out_file_for_raw_data: None
maxPairedFragmentLength: 1000
Traceback (most recent call last):
  File "/home/anaconda3/bin/bamCoverage", line 12, in <module>
    main(args)
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/bamCoverage.py", line 256, in main
    wr.run(writeBedGraph.scaleCoverage, func_args, args.outFileName,
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/writeBedGraph.py", line 171, in run
    bedGraphToBigWig(chrom_names_and_size, [x[3] for x in res], out_file_name)
  File "/home/anaconda3/lib/python3.8/site-packages/deeptools/writeBedGraph.py", line 293, in bedGraphToBigWig
    bw.addHeader(chromSizes, maxZooms=10)
RuntimeError: Received an error while writing the bigWig header
[bwClose] There was an error while finishing writing a bigWig file! The output is likely truncated.

I also tried to use bamCoverage with bam files of which duplicates were not removed. However, it caused the same error. I guess that samtools argument may not be correct, but I do not know what it is.
Would you please help me know what I can try to fix it?

Thank you for your support.

Best

ADD REPLY

Login before adding your answer.

Traffic: 2174 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6