ASHLEYS QC tends to give score <0.3 for libraries with <200k aligned reads, even though they contain good-quality Strand-seq data according to my manual QC. To be clear, this is a relatively minor issue and I will continue to happily use ASHLEYS QC as is, but I will probably do manual QC from now on for low coverage libraries. Overall---thanks for making such a great QC tool!
To confirm that this issue wasn't a quirk of the particular library pool, I also looked at the 28 libraries with between 5k and 200k aligned reads from a completely separate library prep experiment. Half of them (14) scored <0.5 with ASHLEYS QC but looked perfectly fine to me.
As an aside, it could also be argued that we don't want libraries in the 100-200k range for aligned reads anyway (most of the errors below are for libraries in that range). I personally think they are valuable, because they still show SCEs and contribute reads towards inversion calls and phasing.
The command:
ashleys.py -j $1 features -f ./output/bam -w 5000000 2000000 1000000 800000 600000 400000 200000 -o ./output/bam/features.tsv
ashleys.py predict -p ./output/bam/features.tsv -o ./output/bam/quality.txt -m scripts/tools/svc_default.pkl
Manual vs automated QC for 79 libraries. I did this blind to the ASHLEYS QC scores on libraries with 75 bp reads:
Some example of disagreements:
| ASHLEYS QC score |
Manual QC |
Aligned reads |
| 27.72% |
good |
140946 |
| 2.53% |
good |
123526 |
| 2.63% |
good |
136970 |
| 2.07% |
good |
153016 |
| 9.91% |
good |
127418 |
| 2.37% |
good |
124374 |
| 14.97% |
good |
140564 |
| 4.82% |
good |
121376 |
| 11.46% |
good |
427612 |
| 20.49% |
good |
153288 |
BreakpointR plots for two example libraries that I thought were good but ASHLEYS QC did not:
ASHLEYS QC tends to give score <0.3 for libraries with <200k aligned reads, even though they contain good-quality Strand-seq data according to my manual QC. To be clear, this is a relatively minor issue and I will continue to happily use ASHLEYS QC as is, but I will probably do manual QC from now on for low coverage libraries. Overall---thanks for making such a great QC tool!
To confirm that this issue wasn't a quirk of the particular library pool, I also looked at the 28 libraries with between 5k and 200k aligned reads from a completely separate library prep experiment. Half of them (14) scored <0.5 with ASHLEYS QC but looked perfectly fine to me.
As an aside, it could also be argued that we don't want libraries in the 100-200k range for aligned reads anyway (most of the errors below are for libraries in that range). I personally think they are valuable, because they still show SCEs and contribute reads towards inversion calls and phasing.
The command:
Manual vs automated QC for 79 libraries. I did this blind to the ASHLEYS QC scores on libraries with 75 bp reads:
Some example of disagreements:
BreakpointR plots for two example libraries that I thought were good but ASHLEYS QC did not:
