Warning in CuffDiff when using novel transcripts from CuffMerge as input

[komalsrathi]

Hi everyone,

I am doing a differential gene expression test on RNASeq data from 64 Human Heart samples as follows:

cufflinks --library-type fr-firststrand -p 2 -o cufflinks_out <bam_file>

This gave me 64 transcripts.gtf files, one per sample. I created a list of the file names and saved it to assembly_list.txt

I then downloaded three GTF files from Gencode v19 : 1. Protein Coding Genes 2. Long Non-coding RNAs and 3. Pseudogenes. I merged the three gtf files, extracted out just the entries matching exon and CDS in the third column and used it as input for cuffmerge as follows.

cuffmerge -o cuffmerge_out -g gencode.v19.protein.ncRNA.pseudos_exonCDS.gtf -s hg19.fa -p 25 assembly_list.txt

and then I extracted the novel transcripts matching class_code="u":

chr1 Cufflinks exon 16971271 16972173 . + . gene_id "XLOC_000205"; transcript_id "TCONS_00001942"; exon_number "1"; oId "CUFF.386.1"; class_code "u"; tss_id "TSS739";
chr1 Cufflinks exon 16972864 16972878 . + . gene_id "XLOC_000205"; transcript_id "TCONS_00001942"; exon_number "2"; oId "CUFF.386.1"; class_code "u"; tss_id "TSS739";
chr1 Cufflinks exon 20609243 20609496 . + . gene_id "XLOC_000241"; transcript_id "TCONS_00002229"; exon_number "1"; oId "CUFF.393.1"; class_code "u"; tss_id "TSS855";
chr1 Cufflinks exon 20611093 20611502 . + . gene_id "XLOC_000241"; transcript_id "TCONS_00002229"; exon_number "2"; oId "CUFF.393.1"; class_code "u"; tss_id "TSS855";
chr1 Cufflinks exon 33925677 33925957 . + . gene_id "XLOC_000396"; transcript_id "TCONS_00003679"; exon_number "1"; oId "CUFF.651.1"; class_code "u"; tss_id "TSS1384";
chr1 Cufflinks exon 33926256 33926832 . + . gene_id "XLOC_000396"; transcript_id "TCONS_00003679"; exon_number "2"; oId "CUFF.651.1"; class_code "u"; tss_id "TSS1384";
chr1 Cufflinks exon 56905467 56905577 . + . gene_id "XLOC_000616"; transcript_id "TCONS_00005969"; exon_number "1"; oId "CUFF.1131.1"; class_code "u"; tss_id "TSS2162";
chr1 Cufflinks exon 56910015 56910124 . + . gene_id "XLOC_000616"; transcript_id "TCONS_00005969"; exon_number "2"; oId "CUFF.1131.1"; class_code "u"; tss_id "TSS2162";

and used it in cuffdiff as follows:

cuffdiff -o cuffdiff_out --library-type fr-firststrand -L Control,HF --multi-read-correct --upper-quartile-norm -b hg19.fa --num-threads=20 novels.gtf <control1_bam,control2_bam,...> <hf1_bam,hf2_bam,...>

This is giving me a warning repeatedly and is not moving ahead:

Warning: Your version of Cufflinks is not up-to-date. It is recommended that you upgrade to Cufflinks v2.2.1 to benefit from the most recent features and bug fixes (http://cufflinks.cbcb.umd.edu).
[09:32:46] Loading reference annotation and sequence.
[09:33:06] Inspecting maps and determining fragment length distributions.
Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges. It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.

I have never come across this kind of warning before. Any suggestions/help would be appreciated.

Thanks!