Noi/NOTES/2012-7-6
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Preparation of sheared gDNA for WGBS[edit]
- Ideally Dr. Zhang wants me to prepare WGBS libraries of the trios samples either of HAPMAP CEU or Yoruba. However the whole genome sequencing and RNA sequencing data are available for some samples but not all sample in the trios listed in the GMI grant proposal.
- The links I used to check the whole genome and RNA sequencing data availabilities:
Below table are HAPMAP samples I have processed by capturing with BSPP
Catalog ID | Sex | Family | Relationship | Whole genome sequnces | RNA Seq |
GM10860 | Male | 1362 | father | no | no |
GM11992 | Male | 1362 | paternal grandfather | yes | yes |
GM11993 | Female | 1362 | paternal grandmother | yes | yes |
GM10861 | Female | 1362 | mother | no | no |
GM11994 | Male | 1362 | maternal grandfather | yes | yes |
GM11995 | Female | 1362 | maternal grandmother | yes | yes |
GM12801 | Male | 1454 | father | no | no |
GM12812 | Male | 1454 | paternal grandfather | yes | yes |
GM12813 | Female | 1454 | paternal grandmother | yes | yes |
GM12802 | Female | 1454 | mother | no | no |
GM12814 | Male | 1454 | maternal grandfather | yes | yes |
GM12815 | Female | 1454 | maternal grandmother | yes | yes |
- At the end he told me to include the trios in family 1363 one trios from the table above (I chose family 1362) and the trios from Yoruba (but not the one listed in the grant proposal because the samples were not available)
Sample list[edit]
- Sample preparation to shear DNA at BioGem, UCSD
- Prepare DNA in total volume either 50ul or 130ul in H2O or EB buffer is fine (I used 50ul following Alice's suggestion and her set up).
- Size ~400bp
Labeled on the tube | Sample IDs | Family | Relationship | Population | Position in HAPMAP PT01 plate | Conc. (ng/ul) | Volume for 1000ng (ul) | H2O | Total volume (ul) |
1 | GM12891 | 1463 | Maternal grandfather | CEU | G8 | 250.00 | 8.00 | 42.00 | 50.00 |
2 | GM12892 | 1463 | Maternal grandmother | CEU | B12 | 250.00 | 8.00 | 42.00 | 50.00 |
3 | GM10861 | 1362 | Mother | CEU | A9 | 250.00 | 8.00 | 42.00 | 50.00 |
4 | GM11994 | 1362 | Maternal grandfather | CEU | G9 | 250.00 | 8.00 | 42.00 | 50.00 |
5 | GM11995 | 1362 | Maternal grandmother | CEU | G7 | 250.00 | 8.00 | 42.00 | 50.00 |
6 | GM18506 | Y009 | Child | YRI | Tube from Dr. Zhang | 190.00 | 10.52 | 39.48 | 50.00 |
7 | GM18507 | Y009 | Father | YRI | Tube from Dr. Zhang | 263.00 | 7.60 | 42.40 | 50.00 |
8 | GM18505 | Y009 | Mother | YRI | Tube from Dr. Zhang | 218.30 | 9.17 | 40.83 | 50.00 |
Follow up the set up for Covaris shearing (From BioGem and Alan's note: [[1]] Target BP 400 Tube Micro Duty Factor 10% Peak Incident Power (w) 140 Cycle per Burst 200 Time (secs) 55 Volume 50ul Temperature (c) 7 Intensifier Yes
Technique of pipeeting DNA samples from 0.2mL tube to microtube (purchase at BioGem) (suggested by Alice) Since Microtube can not be uncapped, I need to used p200 pipette transfer DNA to microtube by puncture the rubber on the top of the tube. I need to check if the top of the tube is clean to prevent contamination After shearing sheared DNA should be transfered to a clean 0.2mL tube. This time Alice suggested to used p10 pipette instead of p200 to transfer DNA (5 times of pipeting for each sample) due to the pressure issue. Don't forget to spin the tube before transferring DNA If the samples are not process within a week, the sheared DNA should be stored in -20C