Low mapping after Cufflinks assembly
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8 days ago


I am assembling with a reference genome using the BAM files obtained with STAR from 2 samples against the reference genome "MG3_CGAv1.fa". For this I have used the Cufflinks program following the pipeline below.

The assembly I get (transcripts_consensus.fa) seems to be good, with a Completeness (BUSCO analysis) of 95% and a large number of CDS detected. However, when I map the reads against the consensus transcriptome obtained with Cufflinks using the RSEM program, less than 10% map (evidently there is some problem). If I map my reads against that generated transcriptome with STAR, again, almost none of them map.

I thought that I could be indicating the strand specificity wrong, but the libraries were built following the "TruSeq Stranded mRNA Library Prep Kit (dUTP protocol)" so I specified that the type of library is fr-firststrand in Cufflinks and in RSEM I map as stranded reverse (even indicating non-stranded the result does not change).

Do you see something in the script that could generate this later problem in the RSEM quantification step?


module load cufflinks
module load samtools

# Create a list file with the names of the sorted BAM files
ls *.sorted.bam > sorted_bam_list.txt

# Define the variable bam_list

# Iterate over each file in the list
while IFS= read -r bam_file; do
    # Extract the base filename of the file (without the extension)
    sample_name=$(basename "$bam_file" .sorted.bam)

    # Create a unique output directory for each sample

    # Create the output list file for Cuffmerge
    echo "$output_dir/transcripts.gtf" >> gtf_list.txt

    #Run Cufflinks
    cufflinks -o "$output_dir" --library-type fr-firststrand -G MG3_CGAv.gtf -b MG3_CGAv1.fasta "$bam_file"

    # Rename the resulting GTF file to a sample specific name
    mv "$output_dir/transcripts.gtf" "$output_dir/transcripts_${sample_name}.gtf"

done < "$bam_list"

# Create a list file with the GTF files produced by Cufflinks
ls -d cufflinks_output*/transcripts*.gtf > gtf_list.txt

# Run Cuffmerge to combine the results
cuffmerge -o cuffmerge_output -g MG3_CGAv.gtf -s MG3_CGAv1.fasta -p 4 gtf_list.txt

# Get the output GTF file from Cuffmerge

# Use gffread to convert the GTF file to FASTA
gffread -w transcripts_consensus.fa -g MG3_CGAv1.fasta $merged_gtf
assembly gffread cuffmerge Cufflinks RSEM • 119 views

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