error in the location of chromosomes (bam files) - ncRNA ref (alignament tophat)... error compatibility with ht-seq count

Question

[call]

Hello

I made an alignment with ncRNA.fasta sequence by Ensembl using tophat. however... in my output the trancript_ID stay in the chr local... So when I tried to run ht-seq count none sequence is found..

Why does it happen? there is a way to change information for chromosome or be compatible with ht-seq?

please someone help me!!!

HWI-7001432L:176:C8DRKANXX:6:2102:9302:3530 pUr1s ENSSSCT00000001325.2 5 1 76M * 0 0 GCCACCGCGGTTCGCGGTTCTAAACTCTCCATCCATTCGCCTCGACTCCGCTTCTCTCCAGACTCCGAGGCTGAGG BGGGGGFGGGGGGGDGDGGEGDGGGGGGGGGEGGGGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCC AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:76 YT:Z:UU NH:i:3 CC:Z:ENSSSCT00000001326.2 CP:i:5 HI:i:0 HWI-7001432L:176:C8DRKANXX:2:1214:8451:68280 353 ENSSSCT00000000705.2 606 1 76M = 788 258 CTAAGTTTTCTTTCTTCTGGTTGGGATTATCATGGGATCCATCTGGCCAAAGGTGGCTGTGGGAAGATGGCACGCT BCCBCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGCGDFGGGGGGGGGGGGGGFGGEGGGGGGGG AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:76 YT:Z:UU NH:i:4 CC:Z:ENSSSCT00000033566.1 CP:i:641 HI:i:0

[WouterDeCoster]

Might be that I missed something, but why don't you just align the sequences (using a splice-aware aligner such as STAR) to the genome instead of to this ncRNA.fasta?

[call]

Hello! I did the align with tophat using the ncRNA.fasta..

[Satyajeet Khare]

Hi,

WouterDeCoster is suggesting to try whole genome fasta for pig. Thats what we do for typical tophat command. Transcript information is provided via GTF file. Best

[WouterDeCoster]

Hello call!

It appears that your post has been cross-posted to another site: http://seqanswers.com/forums/showthread.php?p=200202#post200202

This is typically not recommended as it runs the risk of annoying people in both communities.

Answer 1

[Satyajeet Khare]

You need to use genome fasta file and ncRNA gtf file. You can use ncRNA fasta file provided it has chromosomal locations in headers. You can get genome fasta and ncRNA gtf files from Gencode.

[call]

Thank you for the attention! but my ncRNA fasta there are the chromosomal locations in headers: >ENST00000347977 ncrna:miRNA chromosome:NCBI35:1:217347790:217347874:-1 gene:ENSG00000195671 gene_biotype:ncRNA transcript_biotype:ncRNA

But I dont know why this happed...

I work with pig sequence... so not have information in gencode. because that I did used the ensembl information...

[Satyajeet Khare]

Okay, so the fasta format looks slightly different. Three options.

1. Do you have access to ncRNA GTF file? If yes, try that with tophat.
2. If not, you can make one from fasta file. You may need to build transcriptome to run tophat anyway. My guess is, if you do that you might generate GTF file when building the transcriptome. Use this GTF file with genome in fasta format from same build for tophat command.
3. Perform tophat with genome fasta file and total RNA gtf file and then shortlist ncRNAs from your fasta file.

Best,

[WouterDeCoster]

Most straightforward would be option3!