Problem Bam to Fastq

[hypno]

Hi
I want to create 2 fastq file starting from bam allignment.
I didi this:
samtools view -F 8 acc.bam > new.bam
java -jar ~/picard-tools-1.99/ValidateSamFile.jar INPUT=new.bam OUTPUT=foi.bam
java -jar ~/picard-tools-1.99/SamToFastq.jar INPUT=foi.bam FASTQ=p_R1.fq SECOND_END_FASTQ=p_R2.fq VALIDATION_STRINGENCY=LENIENT


This is the error:
To get help, see http://picard.sourceforge.net/index.shtml#GettingHelp
Exception in thread "main" net.sf.samtools.SAMFormatException: Error parsing text SAM file. Not enough fields; Line 1
Line: ERROR: Read groups is empty
at net.sf.samtools.SAMLineParser.reportFatalErrorParsingLine(SAMLineParser.java:420)
at net.sf.samtools.SAMLineParser.parseLine(SAMLineParser.java:210)
at net.sf.samtools.SAMTextReader$RecordIterator.parseLine(SAMTextReader.java:237)
at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:225)
at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:201)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:687)
at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:665)
at net.sf.picard.sam.SamToFastq.doWork(SamToFastq.java:132)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
at net.sf.picard.sam.SamToFastq.main(SamToFastq.java:121)


How can resolve? Thanks in advance for your help!!

[akshaya.ramesh]

I have never done a direct BAM to fastq, but you could use samtools to get a SAM file from your BAM, and then use picard tools (SamtoFastq) for conversion. One important thing to remember is that the SAM file genearted when you use samtools, does not contain a header. Attach the header to your sam file and then picard tools should work fine.

#bam to sam: samtools view in.bam > out.bam

Add your header for the sam files (out.bam)

eg:
@HD VN:1.0 SO:unsorted
@SQ SN:chr10 LN:135534747
@SQ SN:chr11 LN:135006516
@SQ SN:chr12 LN:133851895
@SQ SN:chr13 LN:115169878
@SQ SN:chr14 LN:107349540
@SQ SN:chr15 LN:102531392
@SQ SN:chr16 LN:90354753
@SQ SN:chr17 LN:81195210
@SQ SN:chr18 LN:78077248
@SQ SN:chr19 LN:59128983
@SQ SN:chr1 LN:249250621
@SQ SN:chr20 LN:63025520
@SQ SN:chr21 LN:48129895
@SQ SN:chr22 LN:51304566
@SQ SN:chr2 LN:243199373
@SQ SN:chr3 LN:198022430
@SQ SN:chr4 LN:191154276
@SQ SN:chr5 LN:180915260
@SQ SN:chr6 LN:171115067
@SQ SN:chr7 LN:159138663
@SQ SN:chr8 LN:146364022
@SQ SN:chr9 LN:141213431
@SQ SN:chrM LN:16571
@SQ SN:chrX LN:155270560
@SQ SN:chrY LN:59373566
@PG ID:bowtie2 PN:bowtie2 VN:2.1.0

New file: out.sam

#sam to fastq: java -jar SamToFastq.jar I=out.sam F=out.fastq

[kmcarr]

Hi
I want to create 2 fastq file starting from bam allignment.
I didi this:

samtools view -F 8 acc.bam > new.bam

This command will output a SAM (text) file not a BAM file and that file will not have a header as akshaya.ramesh alread said. The default output mode for samtools view is SAM; to output to BAM you have to use the '-b' option; you can also use '-o' to define the output file instead of redirecting STDOUT:

samtools view -b -F 8 -o new.bam acc.bam

new.bam now contains only those aligned reads whose mates did not align (that's what you want right, since you used the -F 8 flag?).

java -jar ~/picard-tools-1.99/ValidateSamFile.jar  INPUT=new.bam OUTPUT=foi.bam

ValidateSamFile does not output a new bam file, it simply checks an existing file, leaves it unchanged and prints a report to STDOUT. Using the OUTPUT= option just redirects the report to an output file, in this case an output file named foi.bam.

java -jar ~/picard-tools-1.99/SamToFastq.jar  INPUT=foi.bam FASTQ=p_R1.fq SECOND_END_FASTQ=p_R2.fq VALIDATION_STRINGENCY=LENIENT

Based on the comment immediately above you should now understand why this failed. The file 'foi.bam' is not a BAM (or SAM) file, it is simply the report output by ValidateSamFile.

You should use:

java -jar ~/picard-tools-1.99/SamToFastq.jar  INPUT=new.bam FASTQ=p_R1.fq SECOND_END_FASTQ=p_R2.fq VALIDATION_STRINGENCY=LENIENT