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  • RNA-seq analysis for expression result

    Hello;

    I am trying to get familiar with RNA-seq analysis and want to have an overall work flow with the bundle(bowtie--->tophat--->cufflinks) to analyze my RNA-seq data, but there is no clear answer over the website. The closest discussion to my question was addressed to HTSeq in another thread, which seems irrelevant to my question although it shares some points.
    Now I come to the final step to use cuffcompare to get the expression level of each gene. Here is my work flow:

    1. First I built the index:
    Code:
    bowtie-build TAIR10_chrAll.fa  ./Bowtie-indexes/TAIR10
    2. Next I use tophat to map the reads to the genome:
    Code:
    tophat -r 20 -o ./Result-Tophat-out-20110329 -p 24 TAIR10 s_6_sequence.txt
    3. Then I use cufflinks to get the expression of genes and transcripts:
    Code:
    cufflinks -o Result-cufflinks-20110329tair10c -G TAIR10_GFF3_genesb.gff -r TAIR10_chrAll.fa tophat-out/accepted_hits.bam
    4. Then
    Code:
     cuffcompare -o cuffcompare -r TAIR10_GFF3_genes.gff tophat-out/transcripts.gtf
    Now I got result as following which contains redundant information. What I need is just AT1G01040 as a combination of all the transcript of this gene.

    Code:
    trans_id	bundle_id	chr	left	right	FPKM	FMI	frac	FPKM_conf_lo	FPKM_conf_hi	coverage	length	effective_length	status
    AT1G01040.1	31361	Chr1	23145	31227	1.71887e-08	2.38915e-05	3.08942e-10	0	1.05579e-06	2.10524e-09	6251	6151	OK
    AT1G01040.1-Protein	31361	Chr1	23518	31079	0.00071945	1	1.5685e-05	0.00050699	0.00093191	8.81164e-05	7561	7461	OK
    AT1G01040.2	31361	Chr1	23415	31120	29.3171	1	0.494891	21.6766	36.9575	3.59068	5877	5777	OK
    AT1G01040.1,AT1G01040.1-Protein	31361	Chr1	23518	31079	19.0961	1	0.314152	14.1945	23.9976	2.33884	5730	5630	OK
    AT1G01040.2,AT1G01040.2-Protein	31361	Chr1	23518	31079	3.218e-08	1.68516e-09	5.29679e-10	0	0.000645987	3.94133e-09	5733	5633	OK
    I have the standard gtf file of the genome (Arabidopsis TAIR10)

    Code:
    Chr1	TAIR10	gene	3631	5899	.	+	.	ID=AT1G01010;Note=protein_coding_gene;Name=AT1G01010
    Chr1	TAIR10	mRNA	3631	5899	.	+	.	ID=AT1G01010.1;Parent=AT1G01010;Name=AT1G01010.1;Index=1
    Chr1	TAIR10	protein	3760	5630	.	+	.	ID=AT1G01010.1-Protein;Name=AT1G01010.1;Derives_from=AT1G01010.1
    Chr1	TAIR10	exon	3631	3913	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	five_prime_UTR	3631	3759	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	3760	3913	.	+	0	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	exon	3996	4276	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	3996	4276	.	+	2	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	exon	4486	4605	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	4486	4605	.	+	0	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	exon	4706	5095	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	4706	5095	.	+	0	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	exon	5174	5326	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	5174	5326	.	+	0	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	exon	5439	5899	.	+	.	Parent=AT1G01010.1
    Chr1	TAIR10	CDS	5439	5630	.	+	0	Parent=AT1G01010.1,AT1G01010.1-Protein;
    Chr1	TAIR10	three_prime_UTR	5631	5899	.	+	.	Parent=AT1G01010.1
    and the Tophat output gtf file seems fine too.

    Code:
    trans_id	bundle_id	chr	left	right	FPKM	FMI	frac	FPKM_conf_lo	FPKM_conf_hi	coverage	length	effective_length	status
    Chr1	Cufflinks	transcript	3631	5899	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	3631	3913	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	3996	4276	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; exon_number "2"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	4486	4605	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; exon_number "3"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	4706	5095	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; exon_number "4"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	5174	5326	1	+	.	gene_id "AT1G01010.1"; transcript_id "AT1G01010.1"; exon_number "5"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	transcript	3766936	3768212	1000	-	.	gene_id "CUFF.4957"; transcript_id "AT1G11240.1,AT1G11240.1-Protein"; FPKM "37.6814271783"; frac "0.999315"; conf_lo "25.348853"; conf_hi "50.014001"; cov "4.615128";
    Chr1	Cufflinks	exon	3766936	3767192	1000	-	.	gene_id "CUFF.4957"; transcript_id "AT1G11240.1,AT1G11240.1-Protein"; exon_number "1"; FPKM "37.6814271783"; frac "0.999315"; conf_lo "25.348853"; conf_hi "50.014001"; cov "4.615128";
    Chr1	Cufflinks	exon	3767406	3767520	1000	-	.	gene_id "CUFF.4957"; transcript_id "AT1G11240.1,AT1G11240.1-Protein"; exon_number "2"; FPKM "37.6814271783"; frac "0.999315"; conf_lo "25.348853"; conf_hi "50.014001"; cov "4.615128";
    Chr1	Cufflinks	exon	3767885	3767990	1000	-	.	gene_id "CUFF.4957"; transcript_id "AT1G11240.1,AT1G11240.1-Protein"; exon_number "3"; FPKM "37.6814271783"; frac "0.999315"; conf_lo "25.348853"; conf_hi "50.014001"; cov "4.615128";
    Chr1	Cufflinks	exon	3768088	3768212	1000	-	.	gene_id "CUFF.4957"; transcript_id "AT1G11240.1,AT1G11240.1-Protein"; exon_number "4"; FPKM "37.6814271783"; frac "0.999315"; conf_lo "25.348853"; conf_hi "50.014001"; cov "4.615128";
    Chr1	Cufflinks	transcript	3768724	3769706	1	+	.	gene_id "AT1G11250.1"; transcript_id "AT1G11250.1-Protein"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	3768724	3769706	1	+	.	gene_id "AT1G11250.1"; transcript_id "AT1G11250.1-Protein"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	transcript	3768724	3769706	1	+	.	gene_id "CUFF.4961"; transcript_id "AT1G11250.1,AT1G11250.1-Protein"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	3768724	3769187	1	+	.	gene_id "CUFF.4961"; transcript_id "AT1G11250.1,AT1G11250.1-Protein"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    Chr1	Cufflinks	exon	3769274	3769706	1	+	.	gene_id "CUFF.4961"; transcript_id "AT1G11250.1,AT1G11250.1-Protein"; exon_number "2"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
    The output file combined.gtf should to be what I need according to the manual, but what I got still contains redundant information:

    Code:
    Chr1	Cufflinks	exon	3631	3913	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "1"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	3996	4276	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "2"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	4486	4605	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "3"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	4706	5095	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "4"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	5174	5326	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "5"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	5439	5899	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "6"; oId "AT1G01010.1"; nearest_ref "AT1G01010.1"; class_code "="; tss_id "TSS1";
    Chr1	Cufflinks	exon	3760	5630	.	+	.	gene_id "XLOC_000001"; transcript_id "TCONS_00000002"; exon_number "1"; oId "AT1G01010.1-Protein"; nearest_ref "AT1G01010.1-Protein"; class_code "=";
    How do I ask cuffcompare to output the gene-based expression results ignoring all the alternative transcripts? For example, I only need the expression level (FPKM) for AT1G11240 as a union of all the exons and other alternative transcripts.

    I am aware this post is long, but believe this is a very common question for the first step of RNA-seq analysis. Your reply is highly appreciated!

    Yifang

  • #2
    I'm quite new to RNA-seq analysis, but if I were having the same problem I would just write a very simple Perl script to modify my .gtf file so that each "gene" was only represented once with only the start and end coordinates. In your case it looks like you could just pull out every line with the word "transcript" in the third column. Then run cufflinks and cuffcompare again. This would give you FPKM for the entire gene region, so it would include unspliced transcripts, however I'm not really sure what would happen to the spliced transcripts (if they would be counted) that were mapped by tophat.

    Just a suggestion, hope it helps.

    Comment


    • #3
      What do you need cuffcompare for? Couldn't you just take the 'genes.expr' file produced by Cufflinks, if you are interested in quantifying on the known gene level?

      Comment


      • #4
        Re:

        Thanks camg and kopi-o!

        First, I thought of the genes.expr file that may already have what I need, but there is only one sentence in the cunfflinks manual to describe this file. It is hard for me to tell which isoform is the "known gene", and what I want to get a union FPKM of all the isoforms. I was wrong to use the cuffcompare for this purpose!

        However, in the cufflinks' genes.expr file I have following isoforms for one gene e.g AT1G01100:

        Code:
        CUFF.55	31366	Chr1	47704	49166	27.6435	17.0802	38.2069	OK
        AT1G01100.2	31367	Chr1	50074	51199	5.11165	2.05821	8.1651	OK
        AT1G01100.1	31367	Chr1	50089	51108	18.7271	4.20713	33.2471	OK
        AT1G01100.4	31367	Chr1	50089	51187	10.812	2.90036	18.7236	OK
        AT1G01100.3	31367	Chr1	50089	51197	0.10237	0.0695312	0.135209	OK
        CUFF.65	31367	Chr1	50283	50954	3.03936	2.33326	3.74547	OK
        ......
        In the reference GTF file (TAIR10) I noticed the gene_id is marked by gene at the third column of the file. For example for AT1G01100:

        Code:
        Chr1	TAIR10	gene	50075	51199	.	-	.	ID=AT1G01100;Note=protein_coding_gene;Name=AT1G01100
        Chr1	TAIR10	mRNA	50090	51108	.	-	.	ID=AT1G01100.1;Parent=AT1G01100;Name=AT1G01100.1;Index=1
        Chr1	TAIR10	protein	50284	50954	.	-	.	ID=AT1G01100.1-Protein;Name=AT1G01100.1;Derives_from=AT1G01100.1
        Chr1	TAIR10	five_prime_UTR	51064	51108	.	-	.	Parent=AT1G01100.1
        Chr1	TAIR10	exon	51064	51108	.	-	.	Parent=AT1G01100.1
        Chr1	TAIR10	five_prime_UTR	50955	50963	.	-	.	Parent=AT1G01100.1
        Chr1	TAIR10	CDS	50883	50954	.	-	0	Parent=AT1G01100.1,AT1G01100.1-Protein;
        Chr1	TAIR10	exon	50883	50963	.	-	.	Parent=AT1G01100.1
        Chr1	TAIR10	CDS	50419	50631	.	-	0	Parent=AT1G01100.1,AT1G01100.1-Protein;
        Chr1	TAIR10	exon	50419	50631	.	-	.	Parent=AT1G01100.1
        Chr1	TAIR10	CDS	50284	50337	.	-	0	Parent=AT1G01100.1,AT1G01100.1-Protein;
        ......
        I believe the first row gene is the longest transcript, and I assume I should use this one as the gene identifier of my reference.

        It seems none of the output file is matching the longest transcript identifier, or it is hard to say which one is matching the identifier of the reference. So that the union of all the isoform-counts is not there. As camg mentioned, I thought to script a piece of perl code to subtract the information of my interest, which seems not working.
        Also I thought of making another reference GTF file only containing the identifiers (i.e. entries marked gene at the third column), then many genes will be excluded for Arabidopsis (from 35386 to 28775). Did I miss anything to ask cufflinks to do the right thing for me, kopi-a?

        Or,
        Code:
        is it relevant to make a new reference GTF file that only contains the longest transcript for cufflinks?
        I have been struggling to sort this out.Thank you guys for the discussion and I hope to make sure what I am doing is correct.
        Thank you again!

        Yifang
        Last edited by yifangt; 03-30-2011, 05:49 PM.

        Comment


        • #5
          OK, I see your problem. I have only run Cufflinks on human, mouse and rat and for those, the genes.expr file contains what you would expect. In your case, I'm afraid there is no solution but to roll up your sleeves and start scripting.

          Comment


          • #6
            Hi yifangt,

            Do you understand what is the meaning of "FPKM "0.0000000000";" that shown in Cufflink's result?
            I don't really get the exact meaning of "FPKM "0.0000000000";"
            Is it mean that the expression level of that particular gene is very low or no express at all in the sample?
            Thanks for advice

            Comment


            • #7
              Cuffcompare GTF problem

              Dear all.

              I'm woriking with RNASeq in bacteria. After using cuffcompare with a SAM file generated by TopHat (where the FLAG field is 0 or 16, so the reads are not paired-end and not strand-specific), I have the next problem:

              1) I observe in the GTF file generated by cuffcompare that all the EXON are in the same strand (+). In the transcript.gtf used by this application, that were generated by cuffllinks, I can find the all three possibilities in this column ("+", "-", "."). You can see in the second point the command used to execute the cuffcompare, and in the third point the first lines of the to file type used in this command.


              2) The command used is:

              Code:
              cuffcompare -C -o name -r anottated_reference_file_ENSEMBLE.gtf transcripts_1.gtf transcripts_2.gtf transcripts_3.gtf

              3) In the netx lines you can see the head of these three files: anottated_reference_file_ENSEMBLE.gtf, transcripts_1.gtf and name.


              • anottated_reference_file_ENSEMBLE.gtf (generated by gffread using the gff provided in GFF database)


              Code:
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    483    2027    .    +    .    ID=NC_008463.1:dnaA;geneID=NC_008463.1:dnaA
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    483    2027    .    +    .    Parent=NC_008463.1:dnaA
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    2056    3159    .    +    .    ID=NC_008463.1:dnaN;geneID=NC_008463.1:dnaN
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    2056    3159    .    +    .    Parent=NC_008463.1:dnaN
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    3169    4278    .    +    .    ID=NC_008463.1:recF;geneID=NC_008463.1:recF
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    3169    4278    .    +    .    Parent=NC_008463.1:recF
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    4275    6695    .    +    .    ID=NC_008463.1:gyrB;geneID=NC_008463.1:gyrB
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    4275    6695    .    +    .    Parent=NC_008463.1:gyrB
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    7018    7791    .    -    .    ID=NC_008463.1:PA_00060;geneID=NC_008463.1:PA_00060
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    7018    7791    .    -    .    Parent=NC_008463.1:PA_00060
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    7803    8339    .    -    .    ID=NC_008463.1:PA_00070;geneID=NC_008463.1:PA_00070
              gi|116048575|ref|NC_008463.1|    RefSeq    gene    7803    8339    .    -    .    Parent=NC_008463.1:PA_00070

              • transcripts_1.gtf (generated by cufflinks)


              Code:
              gi|116048575|ref|NC_008463.1|    Cufflinks    transcript    456    6695    1000    +    .    gene_id "CUFF.1"; transcript_id "YP_788159.1_ext"; FPKM "287.9045692143"; frac "1.000000"; conf_lo "283.384211"; conf_hi "292.424927"; cov "204.464643"; full_read_support "yes";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    456    6695    1000    +    .    gene_id "CUFF.1"; transcript_id "YP_788159.1_ext"; exon_number "1"; FPKM "287.9045692143"; frac "1.000000"; conf_lo "283.384211"; conf_hi "292.424927"; cov "204.464643";
              gi|116048575|ref|NC_008463.1|    Cufflinks    transcript    483    2027    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788156.1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000"; full_read_support "yes";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    483    2027    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788156.1"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
              gi|116048575|ref|NC_008463.1|    Cufflinks    transcript    2056    3159    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788157.1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000"; full_read_support "yes";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    2056    3159    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788157.1"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
              gi|116048575|ref|NC_008463.1|    Cufflinks    transcript    3169    4278    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788158.1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000"; full_read_support "yes";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    3169    4278    1    +    .    gene_id "CUFF.1"; transcript_id "YP_788158.1"; exon_number "1"; FPKM "0.0000000000"; frac "0.000000"; conf_lo "0.000000"; conf_hi "0.000000"; cov "0.000000";
              gi|116048575|ref|NC_008463.1|    Cufflinks    transcript    7018    8394    1000    -    .    gene_id "CUFF.2"; transcript_id "YP_788160.1_ext"; FPKM "53.1976417172"; frac "0.838009"; conf_lo "48.003521"; conf_hi "58.391763"; cov "37.780008"; full_read_support "yes";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    7018    8394    1000    -    .    gene_id "CUFF.2"; transcript_id "YP_788160.1_ext"; exon_number "1"; FPKM "53.1976417172"; frac "0.838009"; conf_lo "48.003521"; conf_hi "58.391763"; cov "37.780008";

              • name.combined.gtf (generated by cuffcompare)


              Code:
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    456    6695    .    +    .    gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "1"; gene_name "NC_008463.1:gyrB"; oId "YP_788159.1_ext"; nearest_ref "NC_008463.1:gyrB"; class_code "o";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    483    2027    .    +    .    gene_id "XLOC_000001"; transcript_id "TCONS_00000002"; exon_number "1"; gene_name "NC_008463.1:dnaA"; oId "NC_008463.1:dnaA"; contained_in "TCONS_00000001"; nearest_ref "NC_008463.1:dnaA"; class_code "=";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    2056    3159    .    +    .    gene_id "XLOC_000001"; transcript_id "TCONS_00000003"; exon_number "1"; gene_name "NC_008463.1:dnaN"; oId "NC_008463.1:dnaN"; contained_in "TCONS_00000001"; nearest_ref "NC_008463.1:dnaN"; class_code "=";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    3169    4278    .    +    .    gene_id "XLOC_000001"; transcript_id "TCONS_00000004"; exon_number "1"; gene_name "NC_008463.1:recF"; oId "NC_008463.1:recF"; contained_in "TCONS_00000001"; nearest_ref "NC_008463.1:recF"; class_code "=";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    7011    7463    .    +    .    gene_id "XLOC_000002"; transcript_id "TCONS_00008029"; exon_number "1"; gene_name "NC_008463.1:PA_00060"; oId "CUFF.2.1"; nearest_ref "NC_008463.1:PA_00060"; class_code "x"; tss_id "TSS1";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    7709    8390    .    +    .    gene_id "XLOC_000002"; transcript_id "TCONS_00008029"; exon_number "2"; gene_name "NC_008463.1:PA_00060"; oId "CUFF.2.1"; nearest_ref "NC_008463.1:PA_00060"; class_code "x"; tss_id "TSS1";
              gi|116048575|ref|NC_008463.1|    Cufflinks    exon    8463    10377    .    +    .    gene_id "XLOC_000003"; transcript_id "TCONS_00000005"; exon_number "1"; gene_name "NC_008463.1:PA_00080"; oId "YP_788162.1_ext"; nearest_ref "NC_008463.1:PA_00080"; class_code "=";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    13540    14091    .    +    .    gene_id "XLOC_000004"; transcript_id "TCONS_00000006"; exon_number "1"; gene_name "NC_008463.1:tag"; oId "NC_008463.1:tag"; nearest_ref "NC_008463.1:tag"; class_code "=";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    14236    15123    .    +    .    gene_id "XLOC_000005"; transcript_id "TCONS_00000007"; exon_number "1"; gene_name "NC_008463.1:PA_00120"; oId "NC_008463.1:PA_00120"; nearest_ref "NC_008463.1:PA_00120"; class_code "=";
              gi|116048575|ref|NC_008463.1|    RefSeq    exon    15208    15474    .    +    .    gene_id "XLOC_000006"; transcript_id "TCONS_00000008"; exon_number "1"; gene_name "NC_008463.1:PA_00130"; oId "NC_008463.1:PA_00130"; nearest_ref "NC_008463.1:PA_00130"; class_code "=";


              Thanks in advance for your help! :-)


              Rodri

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