Hi,

I use HISAT2 + Cuffdiff to process my 150PE Mouse RNA-seq data.

Recently, I notice there are huge differences in number between compatible_count and total_count in my result. Many genes were underestimated due to zero "compatible count".

I've check the "XS:A:(+-) and it exists in my SAM/BAM files (Below). I visually check alignments with IGV and nothing is strange.

I also have tried different CuffDiff parameters, like –total-hits-norm or --poisson-dispersion, to see any improvements. But parameters didn't work. The only progress is that correct number of total counts was recognized by CuffDiff (Below)

My questions are:

1. What features are taken to consider a read-pair compatible or not by CuffDiff ?
2. Any parameters to increase number of compatible_count?

Thank you very much for your help.

SAM example:

A00123:18:H3MHFD:1:2162:4182:19413  419 1   3054721 1   137M    =   3054721 -137    CTTAGGGGCTTGAGAAAGTTCTCGCCCTCTCACCTGGGGCCTAAGATTGTATCAAGATAACTATGACAATGGCCTGACCTTTAAGGTTCCGCTTCTAACAATCATAAAGCATCCATAGGACTTCCAGGTACCCGCCC   FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFF   AS:i:-5 ZS:i:-5 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:5A131  YS:i:-5 YT:Z:CP XS:A:-  NH:i:3
A00123:18:H3MHFD:1:2162:4182:19413  339 1   3054721 1   137M    =   3054721 -137    CTTAGGGGCTTGAGAAAGTTCTCGCCCTCTCACCTGGGGCCTAAGATTGTATCAAGATAACTATGACAATGGCCTGACCTTTAAGGTTCCGCTTCTAACAATCATAAAGCATCCATAGGACTTCCAGGTACCCGCCC   FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF   AS:i:-5 ZS:i:-5 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:5A131  YS:i:-5 YT:Z:CP XS:A:-  NH:i:3

Default Cuffdiff message:

[12:34:11] Modeling fragment count overdispersion.
> Map Properties:
>   Normalized Map Mass: 749021.00
>   Raw Map Mass: 752500.47
>   Fragment Length Distribution: Empirical (learned)
>                 Estimated Mean: 272.39
>              Estimated Std Dev: 138.57
> Map Properties:
>   Normalized Map Mass: 749021.00
>   Raw Map Mass: 746990.97
>   Fragment Length Distribution: Empirical (learned)
>                 Estimated Mean: 274.52
>              Estimated Std Dev: 131.43
[12:35:12] Calculating preliminary abundance estimates
[12:35:12] Testing for differential expression and regulation in locus.

total-hits-norm

[15:24:31] Modeling fragment count overdispersion.
> Map Properties:
>   Normalized Map Mass: 52162345.85
>   Raw Map Mass: 55210687.49
>   Fragment Length Distribution: Empirical (learned)
>                 Estimated Mean: 273.32
>              Estimated Std Dev: 140.81
> Map Properties:
>   Normalized Map Mass: 52162345.85
>   Raw Map Mass: 49297576.52
>   Fragment Length Distribution: Empirical (learned)
>                 Estimated Mean: 273.60
>              Estimated Std Dev: 131.36
[15:25:33] Calculating preliminary abundance estimates