Kun:LabNotes/ExonomeSeq
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2008 <calendar> name=Kun:LabNotes/ExonomeSeq format=%name/%year-%month-%day date=2008/02/01 view=oneyear </calendar>
2007 <calendar> name=Kun:LabNotes/ExonomeSeq format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>
Probe Design
Some key numbers
- RefExon: 207,671 (compiled from refGene.txt, hg18), total length: 60,879,002bp
- Coding refExons: 166,554, length of coding seq: 29,118,349bp
- Coding refExons + 6bp splicing motifs: 309,387,676bp
- CCDS exons: 165,146; total size (incl. 6bp splicing motif) 30,141,138bp
- miRNA genes: 685, 61,314bp
Dec08 whole exome probe set
- In this round of probe design, constrains for A/C content and A/C homopolymers were included.
- Two slightly different probe sets were designed.
Dec08 set: Probe file
First Round
- First pass (code): Capturing arms do not contain repetitive sequences or SNPs (SNP129)
- Number of probes: 305,124
- Coverage: 83.5% (percentage of bases covered)
- Efficiency: 77.5% (percentage of bases covered once)
- Second pass (code): Allow repetitive sequences or SNPs outside of the terminal 12bp regions of the capturing arms:
- Number of probes: 21,905
- Combined coverage: 89.4%
- Combined efficiency: 76.1%
- Total number of probes that capture unique targets (<90% homology with other sequences): 304,295(RefSeq)/264,245(CCDS).
- Number of targets that are mostly repeats (average 85% repetitive sequences) in the CCDS set: 456
- Number of remaining probes: 263,789
- Number of probes synthesized (H1+H2<=49bp): 258,861 (4,928 need longer oligos); 20065 CCDS genes, 148,649 coding refExons (25,648,790bp, target (incl. splicing motifs): 27,249,854bp; covered by the probes: 24,932,529bp)
- Probes that contain DpnII site in H1/H2: 27,941 (DpnII SS set)
- Probes that do not contain Dpn II site: 230,920 (DpnII DS set)
- Region covered by two probes: 4,670,165bp
- Region outside targets: 7,044,705bp (78% on-target)
- Capturing arms for the 258,861 synthesized probes:RefExonJan09-all-sorted.txt(12.6MB)
- These probes were remapped to the ccdsExon set generated by Athurva. It turned out that 10,192 probes were unmappable. These were the probes that target non-CCDS exons. ccdsExonJan09-all-sorted.txt(12.6MB)
Second round
- Some exons were missing because they were on chrN_random based on the refGene annotation. Athurva has generated a list of ccdsExon and ccdsExonLink based on ccdsGenes downloaded from UCSC. I used this perl script to map all probes in the Jan09 set to ccdsExonLink, and generated a list of the missing ccdsExons (17,111 in total).
- I used getExonProbe1stRound.pl to do the first round of probe design on the list of missing ccdsExons. In this round of design, SNPs or repetitive sequences are not allowed in H1/H2.
- Then I ran getExonProbe2ndRound.pl on the remaining 5578 ccdsExons. SNPs or repetitive seqeuences are allowed outside the 12bp terminal sequences of H1/H2.
- For the 4020 exons that no probes could be designed in the previous two rounds, I ran getExonProbe3rdRound.pl with even less stringent criteria:
- Wider Tm range (48-75C);
- Target length extended to 225bp;
- Allow for more than one probe for small exons.
- After this third design, only 93 exons were still missing.
- A total of 33235 probes were designed for the missing ccdsExons after three rounds.
- 27150/33235 probes capture unique targets (no homologous sequence with >=95% similarity)
- Combining the 33,235 probes with the 258,861 probes ordered in the Jan09 set, a total of 27.3Mb (91%) CCDS exons were covered. 89% CCDS exons were covered by 27,150+258,861 probes.
Third Round
- 89,479 missing exons or gaps were identified using this Perl script.
28Dec08 set: Not used because there isn't a significant improvement in coverage.
- First round (code): Allow repetitive sequences or SNPs outside of the terminal 12bp regions of the capturing arms:
- Number of probes: 311,741
- Coverage: 85.0% (percentage of bases covered)
- Efficiency: 77.2% (percentage of bases covered once)
- First round (code): Allow repetitive sequences or SNPs outside of the terminal 12bp regions of the capturing arms:
- Second round:
- Number of probes:18,439
- Combined coverage: 89.9%
- Combined efficiency: 76.0%
Oct08 whole exome probe set
- Athurva created a list of non-redundant coding exons for all RefSeq genes.
- Athurva has converted my Cpg probe design program for exon probe design. He has made a number of changes including the strategy to search for a optimal set of probes for a large exon. I made additional changes on the codes. Here is the current version of probe design program.
- A 27k probe set(exon102508) was ordered on 10-25-2008. This probe set contains four subset of oligos for different designs/projects.
- Mike Chou ChrX probes(Len >=100bp, TypeIIs): 5904
- Kun Chr21/22 probes (Nicking Enzymes): 2924+5654
- Kun Chr20 probes (TypeIIs): 7334
- MCC_probes(Celso/Feng, Bing Ren's lab): 5184
- Two Agilent probe designs that allow for long linkers were tested:
- TypeIIs design:[Codes] this is similar to the previous designs in that AP1V4/AP2V4 sequences that contain TspRI/DpnII Type IIs cutting sites were used.
- Nicking Enzymes:[Codes] in this design, AP1V4/AP2V4 were replaced with eMIP_CA1_F/eMIP_CA1_R used by Jay Shendure in his eMIP 55k probe design. eMIP_CA1_F/eMIP_CA1_R contain recognition sites for nicking enzymes Nt.AlwI and Nb.BsrDl. Using nicking enzymes will simplified the probe prep step, also the digestion could be more complete and hence the yield could be higher (yet to be validated). The reason I stayed away from the method previously is that a small fraction of probes that contain the cutting sites within H1/H2 are not compatible with this method. My current thinking is that if it does turn out to be simpler and more efficient, we can make the majority of probe using this method, and the incompatible probes with the DpnII/USER method.
- Received the oligo library on 11/20/2008
- Length of some sequences:
- Average captured fragment: 160bp
- Capturing arms: 49bp
- Sequencing adaptors/primers: 115bp
- Average total length: 324bp = 214KDa.
The first probe set: Kun:LabNotes/ExonomeSeq/2007-11-15
- One 55k library received on 12/12/2007, OLID: 018274110001, 10pmole, resuspended in 500ul RT-PCR grade water (new vial) -> 20nM x 500ul -> Take 100ul out to a 1.5ml non-stick tube as the working tube.
- The second probe set: Kun:LabNotes/ExonomeSeq/2007-12-21
- Two Agilent 55k libraries were ordered on 01/04/2008.
- Cosmic3760 0189191
- CpgCes: Cpg28k + CES27k 0189181
Received 01/24/2008. 10pmole, resuspended in 500ul RT-PCR grade water (new vial) -> 20nM x 500ul -> Take 100ul out to a 1.5ml non-stick tube as the working tube.
Overview of experiments
Sequencing library construction
Random primed
- Forward primer (SolFN6):CGACGCTCTTCCGATCTNNNNNN Tm left = 59.20C
- Reverse primer (SolRN6):CATACGAGCTCTTCCGATCTNNNNNN Tm left = 59.45C