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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 โdial-outโ 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, in 384-well plates''': [[Media:Zhang MDA summary-1.pdf| Zhang MDA summary-1.pdf]] [[Media:Zhang Classifier-1.xlsx| Zhang Classifier-1.xlsx]], - Sample vial#1 (AC-718): use the first half of the plate - Sample vial#2 (AC-777): use the whole plate == Sample transfer from 384-well plate to 96-well plate == '''Sample vial#1 (AC-718)''' * There are total 13 samples will be transferred from 384-well plate to 96-well plate: '''Labeling: AC-718-96well_plate1''', in the following positions {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position in AC-718-384-well''' | align="center" style="background:#f0f0f0;"|'''Position in AC-718-96-well_plate1''' |- | F03||A1 |- | J03||B1 |- | L02||C1 |- | M05||D1 |- | N03||E1 |- | P02||F1 |- | P04||G1 |- | P05||H1 |- | P07||A2 |- | N17||B2 |- | A21||C2 |- | J04||D2 |- | K06||E2 |} * CSV input file: [[Media:AC-718-384-well-to-AC-718-96-well_plate1.csv| AC-718-384-well-to-AC-718-96-well_plate1.csv]]. Note: I set up a larger sample volume (15ul) than the actual volume to make sure all liquid is transferred to the new plate * The first eight amplicons from plate 718 will be used for rehearsal (AC-718-96-well_plate1, A1-H1). * For AC-718-96-well_plate1, transfer ~2.5ul A1-H1 to A1-H1 96-well shearing plate for Illumina library (400bp), and transfer ~2.5ul A1-H1 to A2-H2 96-well shearing plate for PacBio library (2kb) * Note: When I try pipetting the sample in 384-well plate with epMotion, the total volume is only 8ul. I then will use only 2.5ul for each library prep. To pipette a very small volume from a tiny well, I need to set the speed to be very low (0.4mm/sec) otherwise there will be some liquid left in the well (~1ul). '''Sample vial#2 (AC-777)''' * We will split the amplicons in this plate into 4 96-well plates {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position in AC-777-384-well''' | align="center" style="background:#f0f0f0;"|'''96-well plate labeling''' |- | A1-H12||AC-777_plate1_A1-H12 |- | I1-P12||AC-777_plate2_I1-P12 |- | A13-H24||AC-777_plate3_A13-H24 |- | I13-P24||AC-777_plate4_I13-P24 |} == epMotion set up == * CSV input files (CSV formatted is recommended to be imported to the protocol, epMotion5075) # [[Media:AC-777_plate1_A1-H12.csv| AC-777_plate1_A1-H12.csv]] # [[Media:AC-777_plate2_I1-P12.csv| AC-777_plate2_I1-P12.csv]] # [[Media:AC-777_plate3_A13-H24.csv| AC-777_plate3_A13-H24.csv]] # [[Media:AC-777_plate4_I13-P24.csv| AC-777_plate4_I13-P24.csv]] * Note: # I prefer to split CSV file into 4 files to prevent mis-transferring of sample to the wrong plates. # I set up a larger sample volume (15ul) than the actual volume to make sure all liquid is transferred to the new plate The tool numbers in the CSV file must match the dispensing tools as follows: 1 - TS_50 2 - TS_300 * Note about DNA transfer from 384-well plate to 96-well plate and from 96well plate to Covaris shearing plate (will be filled later) * 2013/01/14 '''Set up for Covaris shearing 400bp (total volume 50ul in H2O)''' 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 '''Set up for Covaris shearing 2000bp from Chris (PacBio recommendation)''' '''Note:''' This will be adjusted later based on the recommendation of the machine. Need to use "'Mini-tube Clear''' Target Shear Size: 2.0kb Tube: Mini-tube Clear Shearing Volume: 200.0ul (used total volume 50ul) Temp of Chiller: 4C Temp of Bath: 6-8C Duty Cycle: 20 Intensity: 0.1 --> convert to Peak Incident Power (w): 3.5 Cycles per Burst: 1000 Time: 15cycles x 60sec -> this mean it takes 15min to shear 1 sample Amount of DNA in Tube: 2ug-20ug * Transferred sample to striptube and handed to Chris * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-15
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