Noi/NOTES/2013-1-11
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Skin Microbiom Project plans
- From Dr. Zhang note (his email on 11/21/2012):
- Goal: To generate single-cell genome for human skin microbes at the highest quality that we can achieve
To accomplish the goal, we will do (1) Low-pass genome shotgun sequencing of many candidates (2) Deep Illumina PE sequencing of amplicons that have reasonable coverage (3) Generate PacBio reads to improve genome assembly Dr. Zhang' recommendations (1) Identify a list of candidates for low-pass sequencing. Ideally some would be in the NIH top-100 most wanted list. (2) Make Illumina barcoded libraries using epMotion. We can start with a small batch size of 8-16 libraries as a rehearsal, then go up to 48 or 96 libraries. Also there are tens of microgram of DNA for each amplicon. Please take 1/3-1/4 from each well for covaris shearing, and save the rest. Unpurified amplicons can be sheared directly, followed by AMPure bead purification. (3) Perform DSN normalization (Rui), then low-pass sequencing, either MiSeq or one lane of GA IIx. (4) Identify a shorter list of good amplicons for deep sequencing. (5) Use “dial-out” PCR to extract the libraries from the pool created in (3). Alternatively, in (3) keep a fraction of individual libraries prior to pooling. (6) Deep Illumina PE sequencing on GA IIx or HiSeq. (7) Select a short list of genome that we want to further improve, construct PacBio library (Chris), and sequencing one SMRT cell per sample. (8) De novo genome assembly, and genome annotation. Sample summaries from the Bigelow lab: Zhang MDA summary-1.pdf Zhang Classifier-1.xlsx,