Dinh 2011/NOTES/2011-12-27
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Probes Resubsetting[edit]
Sub1-7 (DMR220K)[edit]
- 20,438 probes did not capture with normalization and in multiple capture experiments.
File:Sub1 7 0capture.txt Out of the 20,438: 1,039 captured in the rebalancing experiment so I will put them back (these were removed from the 0 capture file above). The reason for placing them back is because these probes were able to capture and maybe we lost them during probes preparation. Hopefully, we can get these back after re-synthesis.
- 99,054+1,039 probes to resubset
File:Sub1 7 Capture.txt - I wasn't able to upload this file
- For Sub1_7 (DMR220K) I have only 1 capture experiment to resubset with.
File:Sub1 7 efficiencyPlot.png Subsetting scores (normalized counts, not log scores): A > 200 (Subset 1) File:Sub1 7.s1.txt 3544 (#probes) 200=> B > 20 (Subset 2) File:Sub1 7.s2.txt 30213 20=> C > 5.4 (Subset 3) File:Sub1 7.s3.txt 29818 5.4=> D > 0 (Subset 4) File:Sub1 7.s4.txt 24747 E = 0 (Unk) File:Sub1 7.s5.txt 11771 File:Sub1 7 resubset1.png File:Sub1 7 resubset2.png
Exp1-3 (Expansion probes)[edit]
- 32,144 probes did not capture with normalization and in multiple capture experiments.
File:Exp1 3 0capture.txt
- 106,071 probes to resubset
File:Exp1 3 Capture.txt - I wasn't able to upload this file
- For Exp1_3 (Expansion set) I have multiple capture experiment to work with (also in 10 different tissues). I used the average normalized read counts to subset.
File:Exp1 3 efficiencyPlot.png Subsetting scores (normalized counts, not log scores): A > 35 (Subset 1) File:Exp1 3.s1.txt 3428 (#probes) 35 => B > 5 (Subset 2) File:Exp1 3.s2.txt 23849 5 => C > 1.3 (Subset 3) File:Exp1 3.s3.txt 28290 1.3 => D > 0.25 (Subset 4) File:Exp1 3.s4.txt 22440 0.25 >= E (performs poorly) File:Exp1 3.s5.txt 28064 File:Exp1 3 resubset1.png File:Exp1 3 resubset2.png
Notes[edit]
- All reads counts were normalized to per 1 million mapped reads.
- From the rebalancing experiment, I was able to find 8,796 probes involved in off-target capture or inaccurate mapping of reads to probes.
- The off-target capture read counts were between 1-327, with an average of ~ 1.6 reads per 1 million mapped reads.
- It would be helpful to learn more about the reads which were not able to map to any probes, especially for Sub5,Sub6, and Sub7. We know that DMR330K after the first normalization have >90% specificity.
- How did not using Sub6 and Sub7 affected specificity?
- Closer examination of sensitivity and specificity calculation from a few months ago,
The recent PE100 library was from the UPENN set, and was captured with Sub6 and Sub7 => slightly lower specificity The recent PE110 library as well as those in HL094 was not captured with Sub6 and Sub7 => slightly higher specificity, with the exception of PGP1F (may have low DNA quality).
- Thus, DMR330K has high specificity because the off-target capturing probes were also the least efficient probes.
- LC4K probes probably have low specificity but comparable sensitivity because many of the probes are efficient and because of deep sequencing coverage.
' | Total target size | Total mappable bases | Total on target bases | Total target covered >=1X | Sensitivity (%target covered) | Specificity (%mappped base on target) |
LC4K | ||||||
H1-54C | 251797 | 168941334 | 89608268 | 185056 | 0.73494124234999 | 0.53041056252107 |
H1-52C | 251797 | 189704106 | 78915250 | 187795 | 0.74581905264956 | 0.41599125956715 |
PGP1iPS-54C | 251797 | 210319026 | 131291469 | 185174 | 0.73540987382693 | 0.62424912998599 |
PGP1iPS-52C | 251797 | 257710476 | 124280612 | 188753 | 0.74962370480983 | 0.48224897151639 |
PGP1F-54C | 251797 | 301811294 | 159092545 | 188938 | 0.7503584236508 | 0.52712588349991 |
PGP1F-52C | 251797 | 301526024 | 121979569 | 193326 | 0.76778516026799 | 0.40454076693559 |
DMR330K | 0.49742776233768 | |||||
Recent library-free PE110 | 34394092 | 2340766083 | 2254282508 | 24118127 | 0.70122877498845 | 0.96305330309248 |
*Recent library-free PE100 | 34394092 | 1687630717 | 1582327158 | 25050732 | 0.72834404234309 | 0.93760272437611 |
HL094-H1 | 34394092 | 2340766083 | 2254282508 | 24118127 | 0.70122877498845 | 0.96305330309248 |
HL094-PGP1F | 34394092 | 1019718323 | 934477017 | 13888586 | 0.4038073166752 | 0.9164070076242 |
HL094-PGP1iPS | 34394092 | 1900359617 | 1830971383 | 25334157 | 0.73658455644068 | 0.96348678777465 |