error: cuffdiff requires at least 2 sam files

[munnj90]

Hi all,

Sorry I'm aware this has been covered but I've tried the various responses already posted and none of which, unfortunately, have worked. I'm struggling to get the cuffdiff commands to work.

I've aligned my sequence file (bacterial RNA-seq genome) using tophat:

tophat -p 8 -G ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.gff -o ~/John/tophat_rna-seq/psven15/1_psven15 --no-novel-juncs ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa ~/John/RNA-seq/unmodified_seq_files/Sven-WT-8h-minus5PP_S6_R1_001_trimmed.fastq

This appears to have worked fine.

I'm carrying out cuffdiff as described in the protocal, here is my commandline:

cuffdiff -o diff_out6 -b ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa -p 8 -u ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.gff -L WT,RsrR\
~/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam ~/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

I get the error:

You are using Cufflinks v2.2.1, which is the most recent release.
Error: cuffdiff requires at least 2 SAM files

I've tried several iterations of this, minus labels (-L), i orginally had white space character (as is in the paper) behind the "". I've tried with "~/John/..." "./John/..." and "/John/..." for paths.

All paths can be identified with "ls" and .bam files can be converted to .sam files by "samtools view -h".

Any help would be greatly appreciated!

[GenoMax]

Are you comparing only one replicate in this case (one WT and one RsrR) files? Are those two file names separated by a space? Have you tried running the program without the -u option (which is for multi-read correction)? Can you show us output of

$ ls -lh ~/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam
$ ls -lh ~/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

[munnj90]

Thanks for your response GenoMax.

I'm only comparing 1 replicate. Below are the respective $ls -lh:

$ ls -lh ~/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam
$ ls -lh ~/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

-rw-r--r-- 1 ezx11reu bio 1.5M Mar 8 15:15 /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam
-rw-r--r-- 1 ezx11reu bio 4.8M Mar 8 15:59 /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

I just re-ran the files through tophat to make sure they were not interrupted during the processing.

I just tried a few iterations, removing -L, -u and -p also this:

cuffdiff -o diff_out6 -b ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa ~/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam ~/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

Which gives me the same error about requiring 2 sam files.

[GenoMax]

Can you try

$ cuffdiff -o diff_out6 -b ~/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa -L WT,MUT ~/John/tophat_rna-seq/psven15/5_psven15/accepted_hits.bam ~/John/tophat_rna-seq/psven15/7_psven15/accepted_hits.bam

Is this a small dataset or you did not get good alignments? How many sequences did you start with? Can you post the summary of tophat run stats?

[munnj90]

This is a small plasmid found within our strain so i would expect a low relative alignement.

Here are the alignment summaries:

5_psven15 (wt):
Reads:
Input : 5975994
Mapped : 28670 ( 0.5% of input)
of these: 24 ( 0.1%) have multiple alignments (0 have >20)
0.5% overall read mapping rate.

7_psven15 (RsrR):
Reads:
Input : 15786210
Mapped : 104959 ( 0.7% of input)
of these: 93 ( 0.1%) have multiple alignments (0 have >20)
0.7% overall read mapping rate.

Using your suggested command i get the same error message. I also tried it on the whole genome sample to get the same result.

Here is the tophat report WT then mutant:

[2016-03-01 15:20:45] Beginning TopHat run (v2.0.14)
-----------------------------------------------
[2016-03-01 15:20:45] Checking for Bowtie
Bowtie version: 2.2.2.0
[2016-03-01 15:20:45] Checking for Bowtie index files (genome)..
[2016-03-01 15:20:45] Checking for reference FASTA file
Warning: Could not find FASTA file /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa.fa
[2016-03-01 15:20:45] Reconstituting reference FASTA file from Bowtie index
Executing: /gpfs/grace/bowtie2-2.2.2/bowtie2-inspect /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa > /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/5_psven15/tmp/pSVEN.fa.fa
[2016-03-01 15:20:45] Generating SAM header for /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa
[2016-03-01 15:20:45] Reading known junctions from GTF file
[2016-03-01 15:20:45] Preparing reads
left reads: min. length=30, max. length=75, 5975269 kept reads (725 discarded)
[2016-03-01 15:23:05] Building transcriptome data files /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/5_psven15/tmp/pSVEN
[2016-03-01 15:23:05] Building Bowtie index from pSVEN.fa
[2016-03-01 15:23:05] Mapping left_kept_reads to transcriptome pSVEN with Bowtie2
[2016-03-01 15:25:13] Resuming TopHat pipeline with unmapped reads
[2016-03-01 15:25:13] Mapping left_kept_reads.m2g_um to genome pSVEN.fa with Bowtie2
[2016-03-01 15:32:07] Mapping left_kept_reads.m2g_um_seg1 to genome pSVEN.fa with Bowtie2 (1/3)
[2016-03-01 15:33:20] Mapping left_kept_reads.m2g_um_seg2 to genome pSVEN.fa with Bowtie2 (2/3)
[2016-03-01 15:34:26] Mapping left_kept_reads.m2g_um_seg3 to genome pSVEN.fa with Bowtie2 (3/3)
[2016-03-01 15:35:00] Retrieving sequences for splices
[2016-03-01 15:35:00] Indexing splices
Building a SMALL index
[2016-03-01 15:35:01] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2016-03-01 15:35:32] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2016-03-01 15:36:04] Mapping left_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2016-03-01 15:36:20] Joining segment hits
[2016-03-01 15:36:39] Reporting output tracks
-----------------------------------------------
[2016-03-01 15:38:53] A summary of the alignment counts can be found in /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/5_psven15/align_summary.txt
[2016-03-01 15:38:53] Run complete: 00:18:08 elapsed





[2016-03-01 15:43:35] Beginning TopHat run (v2.0.14)
-----------------------------------------------
[2016-03-01 15:43:35] Checking for Bowtie
Bowtie version: 2.2.2.0
[2016-03-01 15:43:36] Checking for Bowtie index files (genome)..
[2016-03-01 15:43:36] Checking for reference FASTA file
Warning: Could not find FASTA file /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa.fa
[2016-03-01 15:43:36] Reconstituting reference FASTA file from Bowtie index
Executing: /gpfs/grace/bowtie2-2.2.2/bowtie2-inspect /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa > /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/7_psven15/tmp/pSVEN.fa.fa
[2016-03-01 15:43:36] Generating SAM header for /gpfs/home/ezx11reu/John/ChIP-seq/reference_genome/indexed_genomes/tophat/pSVEN.fa
[2016-03-01 15:43:36] Reading known junctions from GTF file
[2016-03-01 15:43:36] Preparing reads
left reads: min. length=30, max. length=75, 15785117 kept reads (1093 discarded)
[2016-03-01 15:49:46] Building transcriptome data files /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/7_psven15/tmp/pSVEN
[2016-03-01 15:49:46] Building Bowtie index from pSVEN.fa
[2016-03-01 15:49:47] Mapping left_kept_reads to transcriptome pSVEN with Bowtie2
[2016-03-01 15:56:43] Resuming TopHat pipeline with unmapped reads
[2016-03-01 15:56:43] Mapping left_kept_reads.m2g_um to genome pSVEN.fa with Bowtie2
[2016-03-01 16:17:24] Mapping left_kept_reads.m2g_um_seg1 to genome pSVEN.fa with Bowtie2 (1/3)
[2016-03-01 16:29:26] Mapping left_kept_reads.m2g_um_seg2 to genome pSVEN.fa with Bowtie2 (2/3)
[2016-03-01 16:31:48] Mapping left_kept_reads.m2g_um_seg3 to genome pSVEN.fa with Bowtie2 (3/3)
[2016-03-01 16:33:10] Retrieving sequences for splices
[2016-03-01 16:33:10] Indexing splices
Building a SMALL index
[2016-03-01 16:33:10] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/3)
[2016-03-01 16:33:50] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/3)
[2016-03-01 16:34:27] Mapping left_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/3)
[2016-03-01 16:34:48] Joining segment hits
[2016-03-01 16:35:36] Reporting output tracks
-----------------------------------------------
[2016-03-01 16:41:36] A summary of the alignment counts can be found in /gpfs/home/ezx11reu/John/tophat_rna-seq/psven15/7_psven15/align_summary.txt
[2016-03-01 16:41:36] Run complete: 00:58:00 elapsed

[GenoMax]

Does pSVEN.fa contain only the plasmid? It does not have more than one fasta sequence in it, correct?

[munnj90]

Yes, it is a single fasta sequence containing file.

[munnj90]

I re-build/renamed the reference genomes so that bowtie can find the files. Is this potentially and issue caused by my .gff file? - as far as i'm aware it should be fine (they ask for .gtf). I've put the top 20 lines below.


-------------------
##gff-version 2
# seqname source feature start end score strand frame attributes
pSVEN - source 1 158122 . + . mol_type "genomic DNA" ; strain "ATCC 10712" ; organism "Streptomyces venezuelae"
pSVEN - CDS 481 717 . + . locus_tag "pSVEN15_0001" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MRSIFGAMRSRIPRCAGTIRAAVKAVYDGLFWPRRDGFVWPRLRNGLKWPHLVALSMSTDSEYSPLREAPGIRTARDL" ; product "hypothetical protein" ; transl_table 11
pSVEN - sig_peptide 481 546 . + . locus_tag "pSVEN15_00001" ; inference "ab initio prediction:SignalP:4.1" ; note "predicted cleavage at residue 22"
pSVEN - CDS 736 1278 . - . locus_tag "pSVEN15_0002" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MTDTRDQRPAEATLDFLRSAFPPEWREPALGHEAVADWEQENGVALPEPYRTFIAEISNGSSLGPADDGGLQPLGWLPDSWPDLGPRQPGEPFPLEAAWPWEDDESVDPEDPRIDAAFTKGSIVLGSEDGQSFWLLLTAGPRRGEVWMIADVGAVPAPGDQAWGFEEWVQRWHTGNDWWN" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 1496 1906 . + . locus_tag "pSVEN15_0003" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MTYRFTARAATTEIDPDGYFTEAALAEGEDGSGFILMFMAGEEEPDAQDVELGMDTHCLVTAGQGTAYGCVREAVLDGNVLRISLDPEALESLRLDEGEIEAVIEAPTEDVARFREVLAQVLTYGRADAVPTRLAV" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 2099 2536 . - . locus_tag "pSVEN15_0004" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MTADWYVLIEEDTRTTERADGTELKLHRWMLTGTHRIGPNQAEALEAAEDAALNHLPGILARHARPGEQPARHALLTQDGVWIVLVKQRHRECHIRVTTAQLVHAREEKEAPPKSLKEKLRSAMDGPAPAPKPWSWTPEGKADRT" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 2628 2843 . - . locus_tag "pSVEN15_0005" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MISAMIRPVAGLPTWNHLASSLVLTVLVWAVWYGMQPVYSPCVYFGNHSYEKAVEYGQCDPPKTRVSTWVQ" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 2999 5524 . + . locus_tag "pSVEN15_0006" ; inference "ab initio prediction:Prodigal:2.6" ; inference "protein motif:Pfam:PF03457.8" ; codon_start 1 ; translation "MTAAACALESFATGRILVTVPTLDLLVQTAQAWRAVGHRAPMIAVCSLENDPVLNSLGVRTTTNPVQLALWAGHGPVVVFATYASLVDREDVDAPEGQRKVRGPLEAALTGGERLYGQRMAPFDLAIVDEAHGTAGDLGRPWAAIHDNARIPADFRLYLTATPRILAAPRPQKGADGQEVELATMTDDPNGTYGAWLAELGLSEAIEREILAGFEIDVLEIRDPSPVLGESEEARRGRRLALLQTALLEHAAAWNLRTVMTFHQKVEEAAAFAEKLPETAAELYMNDASDEDLAQAEKLPPSSIDAEFYELDAGRHVPPDRVWSAWLCGDHLVAERREVLRQFANGIDATGRRVHRAFLASCRVLGEGVDITGERGVEAVCFADTRGSQVEIVQNIGRALRLNRDGSTKVARIIVPVFLEPGEDPEDMVASASFKPLVTVLQGLRSHDERMVEQLASRALTSGKRKAHLRRDEDGRIVGAGGAGDGEDQEEGDDTQAAAEAALLHFSSPRNAATIAAFLRTRVYRPESLVWLEGYQALIRWRKENEITGVHAVPYDVEVEVGITKDFPLGRWVHQQRKALRAGELEDRRKRLLDAPEAGMVWEPGEEAWENKLATLRSYRRATGHLAPRQDAVWGEGEAMVPIGQHMANLRRKGGLGKDPKRAAERAAQLAEIDEDWNCPWPLDWQRHYRVLADLVDADGVLPAIEPGVLFEGDDIGRWLERQKNPGTWAQLSTEQQQRLSKLGVQPDQAPSPAPAAAHATKGPSKAQQAFQRGLVALTQWVEREGDRSVPRGHSEQITVDGEAEPVPVKLGVWLSNTKSRRDKLTTEQRAALAALGMKWA" ; product "Helicase associated domain protein" ; transl_table 11
pSVEN - CDS 5773 6675 . + . locus_tag "pSVEN15_0007" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MFKAAEGVGPQTRPLLVFYGLSQAGRAIAAAAVDLKGEDWNLISHGIHASGYHLDFADIEIRTDPAGTAGSFVRLSELLRSPVWGNDTVVRLEEVWDTLPANLQYPLTGRERFTPLYASADSIDGTDFHPLLTLHVGDILDRVVDDGSRTALDEFLQSYPGTAGYEEFARRRMGAGAEPDYVRHHPNAGWLYMHWKMAAETGTKEERLERLSTMTRSYAGHRYFFPAVAGLPRELHPLMAWWAVLYALSMLARYQPAQWANHINVDGSRHAVPIEKILERAMEHLPVLIADTIEEVAAWP" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 6792 7727 . + . locus_tag "pSVEN15_0008" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MLFEQRMYAHVKFLELMAELYENEFEDFFHRLMCLRYPDFLDVRTAGSLGDRSADGLSLHSRKLYACYAPQTVKPDKIRKKFEGDLSGAVTKRNGEFDTFVFVHNDRRGVHPEVTSLLASARDSMPSLRFEQMGTRRLWRECMQLDQMETEDVLRCEIPIKETTFGIGMEDLAPLLKQLQDIRAESNPLMSLPDVSVEKLDFNQIEGADREDLLRGMRQSYLVDAFYAGTRSELEHDEVAEGFRLYYQQVRRDYSDADDVLWQLKMYFLGSAQPRPKVLRAALVVLAHFFERCDIFETPPAGWQPGIGLTA" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 7724 7990 . + . locus_tag "pSVEN15_0009" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MITPTKGIAPDRCLLAVGAQVLLQLDEPRTVSQTWARLKSWRADQAHTSPVSFEWFVLALDILFAMGAVELVQDVLVARSTDAAPSER" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 7968 9728 . + . locus_tag "pSVEN15_0010" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MLRRLSADDARFKTVEFSPGLNLLVADTTSSSAETDSRNSAGKSSVIELIHFLLGAKSAGSLATNKALRHITFSLAMDWPWQDAPVEVRRRGDNPKVVRLSQDVSGVPADTLFTDSQDVELSVEQWNRVIERDLFGLEGDHPGVSGRTLLSFLIRRVSAHGFNEPTRTFSRQAAAEASSNLAYLLGLDWQLVNGYRELNARKATRDQLRKAVNDPVWGRIVGSTADLRGQITLAEAQVERLRAQVAAFQVVPEYERLKERADQVSRRIKQLAQEDVIDQHNLEELQSAVTETTDVEVSYLEPAYRELGVILNDQVRRRFDDVKAFHHSVVRNRRRFLEEEIQELTARLAARRQERADLGEDQARLLRELADGGALEALTALQTALGREEAALGALRHRFDAAQALEASARQITAKSVELQQAVDLDLQERRQQTDEAILLFSRYAQRLYGEGREAYLAIEAGRTSLSITPRIDADDSRGINNMVIFCFDLTLAVLAHRHERGPDFLIHDSHLYDGVDERQVARALALAAEVTEEEHMQYIVTLNTDTLSTAAQRGFDPEPHIRSPRLTDDEEGGLFGFRFKAAGKA" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 9883 10203 . - . locus_tag "pSVEN15_0011" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MHSLRFTEDADETLNRLTVGSKADPAKLKKVNNALARLQANPRHPGLHSHQYENFPGHASEKVWDSYVENHNPSAWRIYWMYGPDERNAAGETISVITVLVIGPHP" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 10210 10512 . - . locus_tag "pSVEN15_0012" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MTAIHEREEFEIVAETEVDSRGRVSLGRAGARPGRRYRVESNPDGVLLLTPVVSIPEREMQVWNDPHLAERIRTGIEQAKAGKTIDRGDFSHYLDEDDED" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 10654 11499 . - . locus_tag "pSVEN15_0013" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MFCGVKQQTSQKPVASSAQTREALLERAARDFAPIRRAFVQKKRDEADRSSRLAEFVTNGDVRGLRAYLFIVAITSAETAESGWSTTLPGMVWARAFGTTEDATPASARTAVTKVLKRLEERRLITYERPKGTWDIQVTLLREDGSLDPYTRPGNQNRDAYLKIPFELWRTGLIDQLGLPALAMLLTASCEPAFFELPTERMKEWYGWSPDTAERGFRQLAKEGVLQVHKTYIAAPLSPTGTAEVNRYVLLPPFIARSARKKAPSMENTVETTGDAQALGH" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 12187 12744 . + . locus_tag "pSVEN15_0014" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MGQIEEGLERALRTRPVPSSTGARLRFLLAAHRGSTRQVAAVLGVSQRTVQRWVTEKPGARRPPGPTQVRAIEEAVLARWQPRVRARRRAQAEAEGFVFHTRARFGFAAAAGSSDDPRVRWITQDLPGEVARELFAARDAGAGEQQQTVILARALGHAYFREWGRRAHGLHIAFGDIEFADFSIG" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 12777 13856 . - . locus_tag "pSVEN15_0015" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MVIDRHDDVIHTRTALAAHHLPSGRITLHPGPGTTSDTGLAHDLLAALGKPPLLTGRFPAGRQPAWEAAAAWMTALPVTRLTVLRAHRLTTRRTMRLLELRALTGIHLTLVCHRPHLPAALHQALQTADYSLTTDLDAACRHYYGTPVTEPPLADEPAGPTGRWLTLPALERLISYDSPRPCTDPCTPPPITWRHRPPPVPLTAHTAQRVAQRLHAATAHPRLAAAIASALFTGTSLQQLATARPRDYDTAAATLALHDRARYTDGCAAHPVPPWASVFLQAAACFTRLVSGEDQELLAAPGDRVHLLCVAETAKLRPPQPPAACRKAPVGRVEWDWRERQEAERYEAVPTSRVRPSRR" ; product "hypothetical protein" ; transl_table 11
pSVEN - CDS 13903 14646 . - . locus_tag "pSVEN15_0016" ; inference "ab initio prediction:Prodigal:2.6" ; codon_start 1 ; translation "MTAATYQYVDLPDASVVTTRALLTARENITDTVAARAMMCIHGGAGFGKTLAVNTCLRALEPGEDVRKITFRARPTARAVRYELFTALDLAGEPPRHPSEFDRLLKTALAERPRTFLVDEAQWLNGEAFEYFRYLWDEPSTQLAIIFVGGEGCHTVLRREPMLSSRIFIWQHFTRLTPSEVLDVIPLFHPIWADAAPEDIAFADQHAAHGNFRAWAQLTAHTRTALARTGRPRVDQELLRWAFSRLA" ; product "hypothetical protein" ; transl_table 11

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

[GenoMax]

Can you try removing the two track definition lines at the top and see if that works?