how to convert *.gbff to gff file

[Xiaofei Wang]

Dear folks,

Do you folks have this experience/problme before?

 I tried to used the annotation file (annotated by NCBI PGAP) to do alignment by tophat2. Here is what I did:

1. download  LKIW01.1.gbff.gz http://www.ncbi.nlm.nih.gov/Traces/wgs/LKIW01?

2. using bioperl code to transfer it to gff3 format

$ perl /tools/cluster/6.2/perl/5.16.3/bin/bp_genbank2gff3.pl -r LKIW01.1.gbff -out stdout > Cv017_ncbi.gff3

3. get rid of the sequence line at the bottom of "Cv017_ncbi.gff3" and rename it to Cv017_ncbi_reHeader.gff3

4. change the name of scaffolds in download fasta file "LKIW01.1.fsa_nt.gz", which has been uncompressed and renamed so that the 1st column in the annotation file (Cv017_ncbi.gff3) uses the exact same chromosome/contig names (case sensitive) as shown by the bowtie2-inspect command above.

But I always got this error below. 

Here is the code that I used for tophat2:

$ tophat2 --no-novel-juncs -o .  --library-type fr-secondstrand -G ../../ref/Cv017_ncbi_reHeader.gff3 ../../ref/Cv017_ncbi_reHeader ss_JRC06_R1.fq

I think it is problem with the annotation file because it seems wok well if I don't use the option of "-G" in above code (I have not receive the output while I wrote this but it is running normally). Do you people have this problem before?

[2016-07-14 15:15:09] Beginning TopHat run (v2.0.10)

-----------------------------------------------

[2016-07-14 15:15:09] Checking for Bowtie

  Bowtie version:  2.1.0.0

[2016-07-14 15:15:09] Checking for Samtools

Samtools version:  0.1.18.0

[2016-07-14 15:15:09] Checking for Bowtie index files (genome)..

[2016-07-14 15:15:09] Checking for reference FASTA file

[2016-07-14 15:15:09] Generating SAM header for ../../ref/Cv017_ncbi_reHeader

[2016-07-14 15:15:09] Reading known junctions from GTF file

Warning: TopHat did not find any junctions in GTF file

[2016-07-14 15:15:09] Preparing reads

 left reads: min. length=15, max. length=51, 19314301 kept reads (3 discarded)

Warning: short reads (<20bp) will make TopHat quite slow and take large amount of memory because they are likely to be mapped in too many places

[2016-07-14 15:17:56] Building transcriptome data files ./tmp/Cv017_ncbi_reHeader

[2016-07-14 15:17:56] Building Bowtie index from Cv017_ncbi_reHeader.fa

[FAILED]

Error: Couldn't build bowtie index with err = 1

 

Here are some lines in Cv017_ncbi_reHeader.fa for the scaffolds names:

$ grep '>' Cv017_ncbi_reHeader.fa | head -2

>LKIW01000001

>LKIW01000002

OR

$ bowtie2-inspect --names Cv017_ncbi_reHeader | head 

LKIW01000001

LKIW01000002

LKIW01000003

LKIW01000004

LKIW01000005

LKIW01000006

LKIW01000007

LKIW01000008

LKIW01000009

LKIW01000010​

Here are some lines in Cv017_ncbi_reHeader.gff3

$ head -2 Cv017_ncbi_reHeader.gff3

LKIW01000001 GenBank CDS 1 659 . - 1 ID=Cv017_00005;Dbxref=GI:973444192;Name=Cv017_00005;Note=Derived by automated computational analysis using gene prediction method: Protein Homology.;codon_start=1;inference=EXISTENCE: similar to AA sequence:RefSeq:WP_011135967.1;product=pathogenicity island 1 effector protein;protein_id=KUM05732.1;transl_table=11;translation=length.220

LKIW01000001 GenBank gene 1 659 . - 1 ID=Cv017_00005.gene;Alias=Cv017_00005;Name=Cv017_00005

Thanks a lot!

Best,

Xiaofei