Hi all,
I know this question was discussed lot. I went through almost all the questions but im not able find the answer for my question. I have a paired end ribo depleted total RNA seq. Post qc everything was great. Then I mapped at the genome level using STAR.
STAR --runMode alignReads --genomeDir $SCRATCHDIR/star_maping/indexed_files/star_index \
--readFilesCommand zcat --readFilesIn Read_1.fastq Read_2.fastq\
--outSAMtype BAM SortedByCoordinate \
--limitBAMsortRAM 16000000000 \
--outFilterMultimapNmax 1 \
--sjdbGTFfile /GRCh38.86.gtf \
--outFilterMatchNmin 30 \
--runThreadN 6 \
--quantMode GeneCounts \
--outFileNamePrefix /output/$my_name
After mapping for a BAM
Number of input reads | 248315401
Average input read length | 126
UNIQUE READS:
Uniquely mapped reads number | 229793775
Uniquely mapped reads % | 92.54%
Then I used featureCounts to extract the read count using the following parameter.
featureCounts -F GTF -t exon -g gene_id -s 2 -d 50 -D 800 -B -T 6 -a GRCh38.86.gtf -o ${my_name}_feature_counts.txt sample.bam
When I had look at the summery files I got really lost.
Process BAM file /scratch.ssd/praveenkumar/job_19723707.meta-pbs.metac ... ||
|| Paired-end reads are included. ||
|| Assign reads to features... ||
|| Total reads : 459587550 ||
|| Successfully assigned reads : 132295130 (28.8%) ||
Summery file.txt
Assigned 132295130 Unassigned_Ambiguity 10281242 Unassigned_MultiMapping 0 Unassigned_NoFeatures 317011178
Unassigned_Unmapped 0
Unassigned_MappingQuality 0
Unassigned_FragmentLength 0
Unassigned_Chimera 0
Unassigned_Secondary 0
Unassigned_Nonjunction 0
Unassigned_Duplicate 0
How come the featureCounts reads are almost twice as star assigned unique reads. Is there any way to find?
Thanks in advance
1 answer
Featurecounts total reads are exactly twice as 229793775*2 = 459587550.
Since you have a paired-end library you should include the following two parameters in your featureCounts command line.
-p If specified, libraries are assumed to contain paired-end
reads. For any library that contains paired-end reads, the
'countReadPairs' parameter controls if read pairs or reads
should be counted.
--countReadPairs If specified, fragments (or templates) will be counted
instead of reads. This option is only applicable for
paired-end reads. For single-end data, it is ignored.
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please review all the questions you asked during the last seven years and validate them if someone gave the correct answer. Thanks.
when there is a discrepancy in counts, and the counts are close to be double (or half) then the first thing I think of is that the paired-end reads are perhaps reported/counted in single mode
The report from star is Uniquely mapped reads number | 229793775. How star reports for the paired end library. Featurecounts total reads are exactly twice as 229793775*2 = 459587550. can you please verify my featurecounts parameter. Whether i made any mistake or not. For example featurecount reported me paired-end NO but in the next line it says