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Old 01-25-2016, 11:32 AM   #3
gtduarte
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Location: Brazil

Join Date: Jan 2016
Posts: 5
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Hello dan, thanks for replying. I tried what you suggested, running cufflinks with the -g option instead of -G, but unfortunately it didn't work:

$ cufflinks -o cuff_g -g ~/path_to/A_thaliana.TAIR10.30.gtf -b ~/path_to/A_thaliana.TAIR10.30.fa myfile_sorted.bam

Indeed the resulting transcripts.gtf was a bit different from the previous one, for instance:

-> with -G switch:

1 Cufflinks transcript 11649 13714 1000 - . gene_id "gene:AT1G01030"; transcript_id "transcript:AT1G01030.1"; FPKM "0.4354775887"; frac "1.000000"; conf_lo "0.217739"; conf_hi "0.653216"; cov "1.257353";

-> with -g switch:

1 Cufflinks transcript 11649 13714 1000 - . gene_id "AT1G01030"; transcript_id "AT1G01030.1"; FPKM "0.4330886861"; frac "1.000000"; conf_lo "0.216544"; conf_hi "0.649633"; cov "1.250649"; full_read_support "yes";

However, those bam_errors continue to appear when I run cuffmerge, as before, just as the XLOC values as my gene ids:

Example of the merged.gtf:

1 Cufflinks exon 3631 3913 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000002"; exon_number "1"; gene_name "NAC001"; oId "transcript:AT1G01010.1"; nearest_ref "transcript:AT1G01010.1"; class_code "="; tss_id "TSS1"; p_id "P1";

Nevertheless I run cuffdiff, and there were the XLOCs:

From gene_exp.diff:

test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significant
XLOC_000001 XLOC_000001 NAC001 1:3630-5899 wtmock1 wtaba1 OK 2.82389 5.42847 0.94286 0.938326 0.3329 0.999039 no


Just in case, I checked tophat accepted_hits.bam headers, but apparently it seems fine:

$ samtools view -H wtaba1_sorted.bam

@HD VN:1.0 SO:coordinate
@SQ SN:1 LN:30427671
@SQ SN:2 LN:19698289
@SQ SN:3 LN:23459830
@SQ SN:4 LN:18585056
@SQ SN:5 LN:26975502
@SQ SN:Mt LN:366924
@SQ SN:Pt LN:154478
@PG ID:TopHat VN:2.1.0 CL:/usr/bin/tophat -N 3 --read-edit-dist 4 --read-realign-edit-dist 0 -a 6 --microexon-search -r 150 --mate-std-dev 200 -i 8 -I 10000 --min-segment-intron 8 --max-segment-intron 10000 --b2-very-sensitive /path_to/bowtie_index/A_thaliana.TAIR10.30 myfile1_1_paired.fastq.trim myfile1_2_paired.fastq.trim


Do you have any other clue?

Many thanks again!
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