Noi/NOTES/2013-1-11

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Skin Microbiom Project plans[edit]

  • 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, in 384-well plates: 
 Zhang MDA summary-1.pdf
 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[edit]

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
Position in AC-718-384-well 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: 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
Position in AC-777-384-well 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[edit]

  • CSV input files (CSV formatted is recommended to be imported to the protocol, epMotion5075)
  1. AC-777_plate1_A1-H12.csv
  2. AC-777_plate2_I1-P12.csv
  3. AC-777_plate3_A13-H24.csv
  4. AC-777_plate4_I13-P24.csv
  • Note:
  1. I prefer to split CSV file into 4 files to prevent mis-transferring of sample to the wrong plates.
  2. 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