Is Gene's counts done correctly?
0
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Entering edit mode
2.9 years ago
Nick • 0

Hi everyone, I faced problem during RNA-seq and thinking that I might wrongly do the counting. Please take a look at my result file is it good results for counting? I used the following command

counts <- featureCounts(files=bam.files, annot.inbuilt="hg19")

And got this result

  Load annotation file hg19_RefSeq_exon.txt ... ||
|| Features : 225074 ||
|| Meta-features : 25702 ||
|| Chromosomes/contigs : 52 ||
|| ||
|| Process BAM file C33A_Scrambled_Repl_1.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82815682 ||
|| Successfully assigned alignments : 65832460 (79.5%) ||
|| Running time : 2.16 minutes ||
|| Process BAM file C33A_Scrambled_Repl_2.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82928846 ||
|| Successfully assigned alignments : 64328322 (77.6%) ||
|| Running time : 2.17 minutes ||
|| Process BAM file C33A_Scrambled_Repl_3.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82668210 ||
|| Successfully assigned alignments : 65392168 (79.1%) ||
|| Running time : 2.15 minutes ||
|| Process BAM file C33A_shoct4_Repl_1.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 81137416 ||
|| Successfully assigned alignments : 63713196 (78.5%) ||
|| Running time : 2.11 minutes ||

|| Process BAM file C33A_shoct4_Repl_2.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 83018332 ||
|| Successfully assigned alignments : 65587118 (79.0%) ||
|| Running time : 2.15 minutes ||
|| ||

|| Process BAM file C33A_shoct4_Repl_3.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 83188066 ||
|| Successfully assigned alignments : 65159527 (78.3%) ||
|| Running time : 2.15 minutes ||
|| ||
|| Process BAM file HeLa_Scrambled_Repl_1.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 80346438 ||
|| Successfully assigned alignments : 65994453 (82.1%) ||
|| Running time : 2.09 minutes ||
|| ||
|| Process BAM file HeLa_Scrambled_Repl_2.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82543326 ||
|| Successfully assigned alignments : 66828312 (81.0%) ||
|| Running time : 2.15 minutes ||
|| ||
|| Process BAM file HeLa_Scrambled_Repl_3.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82785110 ||
|| Successfully assigned alignments : 65540550 (79.2%) ||
|| Running time : 2.16 minutes ||
|| ||
|| Process BAM file HeLa_shoct4_Repl_1.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 81527452 ||
|| Successfully assigned alignments : 60717022 (74.5%) ||
|| Running time : 2.08 minutes ||
|| ||
|| Process BAM file HeLa_shoct4_Repl_2.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 82734900 ||
|| Successfully assigned alignments : 67453962 (81.5%) ||
|| Running time : 2.14 minutes ||
|| ||
|| Process BAM file HeLa_shoct4_Repl_3.AddRG.Reorder.Sort.bam... ||
|| Paired-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 80796768 ||
|| Successfully assigned alignments : 65203366 (80.7%) ||
|| Running time : 2.09 minutes
RNA-Seq counts • 841 views
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1
Entering edit mode

If you have doubts about your results, double check them. You can do this by looking for a gene with a few reads (lets say 3 read counts for gene x in sample y). Then look in IGV in the bam file for gene x in sample y and count the reads.

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0
Entering edit mode

Actually, I'm the newbie, so during a rna-seq analysis I faced strange values, so trying to figure out is it my mistake or given bam files are not correct.

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1
Entering edit mode

If you want to know if your bam files are correct, you should rephrase the question (that's not what your question currently is asking). Or tell more about the strange values you are getting.

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