Athurva Gore/LabNotes/2011-9-23
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TO DO[edit]
- DONE - Design LC Sciences Probes - sent probes to Dr. Zhang, Dinh, and Noi
- Low-input Exomes (Athanasia) - analyze data?
- Free space on Genome-Miner, create Shared Folder for HiSeq
- HiSeq run was postponed to Wednesday
- Still need to reformat Ext6T, ideally before September 29
- Package Exome Scripts for Thomson Lab
- Barcoding Padlock Probe libraries - talk to Dinh and Noi
- Low-input Bisulfite Conversion
- Paper with Sergio
- Papers for Doug - ask Dr. Zhang?
- Review GR Manuscript - by September 28
LC Sciences Probes[edit]
- Turned out I made a mistake in probe design yesterday
- Some probes were allowed to have arms longer than 40 bp total
- Redesigned probes for same targets (YX Genes [HORMAD1, ZG16] and Ecker mutation/memory that overlaps with PGP1)
- Obtained 2199 probes that captured at least one CpG and had fewer than 3 CpGs in probe arms
- Removed targets with high numbers of probes required (due to C's in arms and large size)
- Also removed 9 additional probes to make the number to order exactly 4000
- Excel Worksheet (for removal of probes): File:OutputFile LC 09232011.xlsx
- Probe Set: File:NM LC Probes Filtered 09232011.txt
- 1820 probes, with 4000 to order due to degeneracy
- Captures 14,228 CpG sites
- Created oligos using script: File:Probe2padlockCpgLibSept11 fixed09232011.txt
- Final oligos: File:NM LC Probes Filtered 09232011.txt.seq
Probe Order[edit]
- Dr. Zhang discovered that only 3918 probes could be ordered, so he deleted 88 probes.
- Enclosed the order form: File:09232011 UCSD ZhangLab.xls
- We need to order primers to amplify and trim these probes
The protocol for making probes would be different (simpler) from our standard one. It is described in this paper, which basically involves PCR, digestion with two nicking enzymes and PAGE size selection: http://arep.med.harvard.edu/pdf/Porreca07.pdf We need to order the two amplification primers (eMIP_CA_1F/R) and two nicking enzymes (Nt.AlwI; Nb.BsrDI, we might have 1-2 tubes in stock). Also the amplification primers after circularization would be different. Please design these primers (CP-FA/CP-RA with Illumina tails and probably with barcodes) and order them before the probes come in.
- Make sure we order these primers early next week.
Low-input Exomes[edit]
- Alice used small numbers of Lymphocytes to perform Exome Capture from Nextera libraries
- Used suppressor oligonucleotides for Nextera adaptor sequences to hopefully increase specificity of capture
- Used very few cells in each step
- Need to generate statistics for each library (D, E, F)
- Currently doing this blind to number of cells in each sample; see what kind of coverage we have
- Mapped data/called consensus sequence using BWA and GATK
- Create pileup files from .fixed.bam files, restricted to CCDS regions
- Counted statistics: File:HL100 Single Exome Statistics 09242011.xlsx
- Need to normalize number of reads and repeat analysis