Thais' notebook BSB genomics

• Sample locations are in the -80oC freezer inventory

  • filter for Owner==Thais
    • BSB_NC_Box Fin clips OGL Buffer
    • BSB_SMC_ME_Box Fin clips (buffer?)
    • BSB_MD_Box fin clips in OGL buffer
    • BSB_NJ_BOX Fin clips in OGL buffer

• Black sea bass shared datasheet

  • First tab: Fin_Clip_Vial_ID is the vial ID
  • Tab: selected for genomics has 64 samples that Thais extracted last March, Thais thinks the extracts are in the boxes in the -80C (even though these boxes say tissue, she thinks they are the extracts).
    • BSB_NC_Box Fin clips OGL Buffer
    • BSB_SMC_ME_Box Fin clips (buffer?)
    • BSB_MD_Box fin clips in OGL buffer
    • BSB_NJ_BOX Fin clips in OGL buffer
  • remaining samples
    • ~20-25 samples from RI in the freezer - Katie thinks this is the box in the -20C
      • we need to check the length data, but we can extract all these
    • Marissa has <10 samples from ME
      • waiting to receive them from Marissa

• Notebook

  • Up to March 12, was troubleshooting
  • Unique_ID is what is used to match to datasheet
  • 20201118 Thais made some changes to the entry 2020-01-BSB-DNA-extraction_summary.md to clarify dilution factors used to measure sample concentrations using Qubit and to provide a separate table listing the 62 extracted samples to be sent out to sequencing facility.
  • 20201118 Thais fixes some typos in the notebooks (diddn't change anything regarding number of samples or DNA quants).

• Sequencing facility contact info:

  • "We request DNA to be sent with volumes >20 ul and concentrations >25 ng/ul as measured by Picogreen or Qubit since Nanodrop will tend to overestimate the amount of double stranded DNA available. However, we input 10 ul of DNA at 10 ng/ul into our GBS library preparation, so DNA >10 ng/ul as measured by Picogreen or Qubit is acceptable. Our library prep rate includes an initial Picogreen QC step, and we can still attempt library prep on sequencing on samples with concentrations <10 ng/ul. We have successfully sequenced samples with initial concentrations <5 ng/ul, however, the results are more variable and we cannot guarantee that the samples will be successful."
  • Email: Evan Forsberg GBS Service Manager 1-202 NHH 312 Church St SE Minneapolis, MN 55455 (612) 624-3177 [email protected]
  • Thais will forward correspondence to Katie and Alan - DONE (TB).
  • Budget: ~$7000-$9000

• Extraction protocol

  • May need to purchase another kit (~$1000)
  • May also needd to purchase quantification kits (Qubit or Picogreen)
  • E.Z.N.A tissue DNA kit (D3396-02, Omega) - tubes (T) method
  • if we use plates, may need to go to Randall's lab
  • Use elution volume less of what they tell you to do (~30uL instead of 100uL for first elution and 70uL out of second elution), stored separately and not combined. Thais checked 2nd elution and not much there.
    • Sara used to use 1/2 the elution volume for her samples, then she combined them

TO DO:

• Sample summary

  • RI samples (southern new england) - data not organized
    • Sample location is our freezer
    • 22 to extract/sequence
  • MA samples from Sept 2020 - where are these stored?
    • Thais gave EtOH vials to them
    • Tommy and Jonathon collected and processed them on the boat
    • In door of standing -20C freezer in Grabowski Lab
    • 20-30: extract as many as we can that are less than 30
  • NJ "conflict" - no date? Fixed with Jon/thais
    • thais has a spreadsheet that came with these samples
  • NC samples - no date? Fixed with Jon/thais
  • ME list - this is a list of vials that Thais sent to Marissa, but never sent back to us
    • Thais says that Marissa didn't enter information to big spreadsheet - Thais made a separate one
    • thais will enter details - June and Nov 2020
    • 15 samples, 9 are smaller than 30cM and 2 marked as YOY
    • Jon will get Marissa to send the finclips

• Sample summary extracted by Thais (62 samples)

  • NJ 17
  • MD 20
  • Seouthern NE 7
  • ME 5
  • NC 13

• Katie

  • Confirm all samples with Jon
  • Get remaining finclips and data from ME - Marissa
  • Quotes from sequencing facility quote ddRAD library prep and sequencing email thread
  • Data quality control - we're not sure if these data are entered yet
    • thais entered vial IDs for Maine samples, because she setn to Marissa
    • thais entered vial IDs for RI/MA samples, because she setn to Jon

• Thais: Go through data in this spreadsheet and transfer to the Black sea bass shared datasheet. DONE (TB)

  • Thais will move this data into the database DONE (TB)
  • Thais thinks we can sequence all of them (all 62 listed above, TB)
  • Notes in right column are based on concentration and volume:
    • plenty: for all samples which we have quantity from Sequencing facility
    • acceptable, could retry: we have enough, but the concentrations are greater than 5 and less than 25ng/uL
    • repeated had to extract twice, some samples we have less than 5

• Alan: extractions

  • extract remaining samples
  • quantify all samples we plan to send to sequencing on plate reader
  • prepare for sequencing requried by sequencing facility
  • help with shipping to sequencing facility