Dinh/Dinh 2012/NOTES/2012-1-11
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Analysis of 9 technical replicates from HL111 (NA12156)[edit]
- Noi performed 9 capture reactions from the same bisulfite converted gDNA, and this library was captured and sequenced in HL111.
- Noi mapped all the data and created the methylation matrix file from the 9 BED files requiring data from at least 7 of the 9 replicates.
- 7 data points is good enough to determine the methylation distribution and variance of a captured site.
- In total, there were : 412,476 sites captured by at least 7 samples.
- However the standard deviation for ~25% of the sites were > 0.1.
- Checking the QC values for this dataset:
rep1 N=9175 r=0.9111 rep2 N=13414 r=0.9292 rep3 N=8385 r=0.9157 rep4 N=16172 r=0.9754 rep5 N=17041 r=0.9738 rep6 N=9770 r=0.9273 rep7 N=15372 r=0.9312 rep8 N=12460 r=0.8802 rep9 N=12389 r=0.8949
- Why does both N and r values vary so much?
- As the average QC drop, which sites become more variable?
- Where these sites variable sites localized?
- We can go back to low quality dataset and mask these variable sites.
DMR330K Resubsetting[edit]
- Continuing from http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh_2011/NOTES/2011-12-27
- I ran this script for generating probes: File:Probe2padlockCpGLibJan11.txt
- Number of padlock generated per subset:
#padlock Subset name (efficiency level) #probes 4334 Exp1_3.s1 (A) 3428 28560 Exp1_3.s2 (B) 23849 33285 Exp1_3.s3 (C) 28290 26490 Exp1_3.s4 (D) 22440 33379 Exp1_3.s5 (E) 28064 7328 Sub1_7.s1 (A) 3544 56748 Sub1_7.s2 (B) 30213 52737 Sub1_7.s3 (C) 29818 43685 Sub1_7.s4 (D) 24747 20620 Sub1_7.s5 (E) 11771 307166 total
- (A,B,C,D,E) are efficiency levels determined from http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh_2011/NOTES/2011-12-27
- Since the change in efficiency from level A to B is much higher than the changes from B to C, C to D, and D to E, probes at level A must be kept separate.
- Although the probes at level E are not very efficient, I have already removed probes that have 0 capture in multiple capture experiments. For Sub1_7, probe placement in E is based on 0 capture in only 1 capture from the re-balancing experiment. For Exp1_3, the cutoff for E is not at 0 capture, so the probes at the top of level E are still good.
- Combine probes at levels B, C, and D (probes in E are not ranked properly since they have 0 capture in 1 experiment)
cat Sub1_7.s2.txt.seq Sub1_7.s3.txt.seq Sub1_7.s4.txt.seq > tmp split -l 55000 tmp DMR220K. 55000 DMR220K.aa (B) 55000 DMR220K.ab (C+D) 43170 DMR220K.ac (D)
- Go into each of the 55K files and make sure that the split is not between variants of the same probe.
- Fill up DMR220K.ac to make 55K:
tail -n 11830 DMR220K.ac > tmp cat DMR220K.ac tmp > DMR220K.ac+
- There are 20,620 probes in level E which can't be ranked. I will add 4334 probes from Exp1_3 level A to this which can be amplified with an alternate reverse primers.
tail -n 2046 Sub1_7.s5.txt.seq > tmp cat Sub1_7.s5.txt.seq tmp > DMR220K.ad ./probe2padlockCpGLibJan11.pl Exp1_3.s1.txt altAP2 cat DMR220K.ad Exp1_3.s1.txt.seq > Mixed_ExpA.DmrE
- DMR220K (Sub1_7) have been subsetted into 3x55K sets. with Mixed_ExpA.DmrE (27K) set.
- Combine probes at levels B, C, D, and E and then split into subsets of 55K for Exp1_3:
cat Exp1_3.s2.txt.seq Exp1_3.s3.txt.seq Exp1_3.s4.txt.seq Exp1_3.s5.txt.seq > tmp split -l 55000 tmp EXP. 55000 EXP.aa (B + C) 55000 EXP.ab (C + D + E) 11714 EXP.ac (E)
- Go into each of the 55K files and make sure that the split is not between variants of the same probe.
- Add 7958 duplicates in EXP.ac (E) with itself to create doubles: 11714 + 7958 (repeats) + 7328 (DmrA) = 27K
./probe2padlockCpGLibJan11.pl Sub1_7.s1.txt altAP2 tail -n 7958 EXP.ac > tmp cat EXP.ac tmp Sub1_7.s1.txt.seq > Mixed_DmrA.ExpE
Ready to order padlock probes[edit]
- DMR330K.s1 File:DMR220K.a.txt.gz 55K
- DMR330K.s2 File:DMR220K.b.txt.gz 55K
- DMR330K.s3 File:DMR220K.c.txt.gz 55K
- DMR330K.s4 File:EXP.a.txt.gz 55K
- DMR330K.s5 File:EXP.b.txt.gz 55K
- DMR330K.s6 File:Mixed ExpA.DmrE.txt.gz 27K
- DMR330K.s7 File:Mixed DmrA.ExpE.txt.gz 27K
- Total 329K padlocks