0-counts for all genes

[NomiCentarix]

Hello,

I am getting 0-counts for all genes in the output GCT files, and also many zeros in the metrics.

Here are the GTF and BAm files are use (google drive link)

My command: rnaseqc Homo_sapiens.GRCh38.98.chr.collapsed.gtf A1_dedup_umi.R2.sorted.bam naseqc_results/

What is wrong with one of my files or what am I doing wrong?

Thanks, Nomi

Edit: Is this because I have 0 high quality reads? I paste my metrics file:

Sample  A1_dedup_umi.R2.sorted.bam
Mapping Rate    1
Unique Rate of Mapped   1
Duplicate Rate of Mapped    0
Duplicate Rate of Mapped, excluding Globins 0
Base Mismatch   0.00360871
End 1 Mapping Rate  0
End 2 Mapping Rate  2
End 1 Mismatch Rate -nan
End 2 Mismatch Rate 0.00360871
Expression Profiling Efficiency 0.795949
High Quality Rate   0
Exonic Rate 0.795949
Intronic Rate   0.0933449
Intergenic Rate 0.0431309
Intragenic Rate 0.889294
Ambiguous Alignment Rate    0.0675756
High Quality Exonic Rate    -nan
High Quality Intronic Rate  -nan
High Quality Intergenic Rate    -nan
High Quality Intragenic Rate    -nan
High Quality Ambiguous Alignment Rate   -nan
Discard Rate    0
rRNA Rate   0.0390048
End 1 Sense Rate    -nan
End 2 Sense Rate    0.955684
Avg. Splits per Read    0.28613
Alternative Alignments  0
Chimeric Reads  0
Chimeric Alignment Rate 0
Duplicate Reads 0
End 1 Antisense 0
End 2 Antisense 50555
End 1 Bases 0
End 2 Bases 166380250
End 1 Mapped Reads  0
End 2 Mapped Reads  1331042
End 1 Mismatches    0
End 2 Mismatches    600418
End 1 Sense 0
End 2 Sense 1090240
Exonic Reads    1059441
Failed Vendor QC    0
High Quality Reads  0
Intergenic Reads    57409
Intragenic Reads    1183687
Ambiguous Reads 89946
Intronic Reads  124246
Low Mapping Quality 0
Low Quality Reads   1331042
Mapped Duplicate Reads  0
Mapped Reads    1331042
Mapped Unique Reads 1331042
Mismatched Bases    600418
Non-Globin Reads    1330951
Non-Globin Duplicate Reads  0
Reads used for Intron/Exon counts   1331042
rRNA Reads  51917
Total Bases 166380250
Total Mapped Pairs  0
Total Reads 1331042
Unique Mapping, Vendor QC Passed Reads  1331042
Unpaired Reads  0
Read Length 125
Genes Detected  0
Estimated Library Complexity    0
Genes used in 3' bias   0
Mean 3' bias    0
Median 3' bias  0
3' bias Std 0
3' bias MAD_Std 0
3' Bias, 25th Percentile    0
3' Bias, 75th Percentile    0
Median of Avg Transcript Coverage   0
Median of Transcript Coverage Std   0
Median of Transcript Coverage CV    0
Median Exon CV  nan
Exon CV MAD nan

[NomiCentarix]

I guess it is because all the reads in my bam file have a mapping quality of 255?

[agraubert]

It looks like your data is unpaired. If this is a single-cell experiment, you can try using getzlab/scrinvex. Otherwise, try running RNA-SeQC with the --unpaired flag to indicate that it should not be expecting read pairs

[NomiCentarix]

Solved! Thank u