[jdjax]

You should check the header information of your bam file. One way to do it is to convert bam to sam using samtools, then check the top portion of the sam files. (e.g., using 'more your.sam'). Let us know what you see in the header.

I am away from the campus network, and do not have access to the server right now. When I back on campus I will post that. Thanks for the help.

[jdjax]

So when looking at the SAM file header line : @HD VN:1.0 SO:coordinate


I also noticed that the rest of the file does not look uniform. The first line there is '@SQ [tab] SN: value [tab] LN: value'. However the following lines are not the same: '@SQ [tab] SN: value LN:value', where there is no tab between the SN and LN value. Through out the header I see some lines that contain '@SQ [tab] SN: value [tab] LN: value' and '@SQ [tab] SN: value LN:value'.

I thought SAM files are supposed to be uniform and have a tab between each column? If I am correct, how do I fix my SAM header?

Thank you again for your help.

[ribozyme]

DZhang,

I did not sort the sam. I am just testing these programs out so I did not use any options for tophat or cufflinks. Tophat made a file accept_hits.bam. I used that file as input for the cufflinks.

My cufflinks command was just: cufflinks accepted_hits.bam

I also want to more descriptive about errors I am recieveing in the hopes of figuring this problem. This is what the error stated:

cufflinks: /usr/lib64/libz.so.1 : no version information available
Warning: BAM header too large
File accepted_hits does not appear to be a valid BAM file, trying SAM
Inspecting reads and determining fragment length distribution.
SAM error on line 2880: CIGAR op has zero length
SAM error on line 3240: CIGAR op has zero length
SAM error on line 3464: CIGAR op has zero length
SAM error on line 5063: CIGAR op has zero length
SAM error on line 30750: CIGAR op has zero length
SAM error on line 51722: CIGAR op has zero length

This continues with increasing line numbers until it reaches the end of the file.
I have also checked /usr/lib64/libz.so.1 and it is in /usr/lib64

libz.so.1 -> libz.so.1.2.3

is what is present in on the server.

Again thanks for your input. I appreciate any help. =)

I am facing the same problem. Any Help Will be appreciated.

[DZhang]

Here is what is said about input file format for cufflinks on its website:

Note the use of the custom tag XS. This attribute, which must have a value of "+" or "-", indicates which strand the RNA that produced this read came from. While this tag can be applied to any alignment, including unspliced ones, it must be present for all spliced alignment records (those with a 'N' operation in the CIGAR string).

The SAM file supplied to Cufflinks must be sorted by reference position. If you aligned your reads with TopHat, your alignments will be properly sorted already. If you used another tool, you may want to make sure they are properly sorted as follows:

sort -k 3,3 -k 4,4n hits.sam

I suspect two things might be the cause: properly sorting the sam/bam file or the CIGAR specification. Tophat will remove these potential problems so I would really recommend giving it a try at least as one of the trouble shooting steps.

[jdjax]

After some serious problem solving we figured out what we need to do to make Bowtie output work with cufflinks.
When working with Bowtie you get a UNsorted SAM file, so you first have to coverted it to a BAM file, then sort the file, then convert it back to a SAM file. Then when you have a sorted SAM file cufflinks does not bring up an error.

I have no idea why this occurs - all I care about is that is works.

Good Luck.

[DZhang]

Hi jdjax,

Thank you for the update and working solution. Did you try "sort -k 3,3 -k 4,4n hits.sam" as suggested by the cufflink website? It it does, it will save you lots of time (and disk space) to convert sam/bam/sam.