This document has been updated on Mon Jun 1 09:14:26 2020 to reflect changes put in place on or about June 1, 2020.

The Process

Trust the Process.

Accounts

We use iLab to handling billing, so make sure you and your PI have active accounts there.

  1. Follow directions at iLab to create accounts
  2. UPenn clients will have dormant accounts that use PennKey credentials, go to iLab to activate your account.
  3. CHOP clients may also have dormant accounts.
  4. See iLab for other institutions.
  5. Make sure the investigator has access to a budget to spend at the NGSC.

We use the NGSC website to handle sample submission and data presentation, so you will also have to have accounts there.

  1. PI creates account if new to NGSC
  2. Investigator (grad student, post doc, staff, etc.) creates account if new to NGSC
  3. Jonathan activates accounts and sends notifications.

Setting Up an Experiment

We are looking to gather enough information from you to determine what the experimental design is and how much will cost.

  1. Investigator fills in Request or Experiment and submits to the NGSC
    • Use the Standard Service Request link under Request Services to make your request.
  2. Jonathan reads request and
    1. generates an estimate
    2. confers with BA, PI, and Investigator to agree on services and charges
  3. BA, PI, and Investigator make entries in iLab to allow NGSC to charge grant
    1. UPenn clients typically use CAMS to set spending permissions which percolate to iLab overnight.
    2. All other clients should load a PO to cover the costs which we have to review and approve.
  4. Jonathan
    1. loads the study
    2. generates sample submission forms
    3. informs investigator about the form

Sample Submission

This section has changed to reflect Phase I pandemic operations.

  1. Investigator downloads, completes, and emails sample submission form to
  2. We review and load samples and informs Investigator that we can accept the samples.
  3. Make an appointment to drop off the samples.
    1. The email confirming that we have loaded the samples will cc the NGSC lab staff.
    2. The lab staff and investigator will set up an appointment for sample drop-off.
    3. We do not have any standard hours for sample drop-off.
  4. Investigator arrives at SCTR 12-161 with samples at the appointment time.
    1. NGSC staff and investigator perform a contactless drop-off observing the current social distancing protocols.
    2. Lab Staff and Investigator match samples with db entries
    3. Lab Staff print labels and apply to tubes
  5. Once the samples arrive they are scheduled for any QC requested.

Quality Checks

These are the possible QC steps we may perform.

Qubit

This is a fluorescence measurement to see how much material is in the sample tube.

  1. perform measurements
  2. load into database.

Agilent BioAnalyzer or TapeStation

This test is a microfluidics-based gel electrophoresis which is used to determine the size distribution of the library or the quality and amount of RNA samples. We are checking to see that the library has the proper size distribution with little or no primer or adapter dimer contamination.

The molarity measurement this test provides for libraries is of limited use, so we augment this test with the Kapa or MiSeq steps below.

  1. perform measurements
  2. place values in core.qc
  3. export BioA data
  4. Lab Staff gives the data to Jonathan
  5. Jonathan generates BioA reports and notifies the Investigator with comments.

Kapa BioSystem Library Quant Kit

This kit uses QRT-PCR to measure the concentration of the library fraction of the sample. To some extend, depending on the experiment and run type, it has been replaced by the MiSeq pre-run sequencing.

Unlike the Qubit or BioAnalyzer steps, the Kape kit assesses only the portion of the sample that contains the Illumina P7 and P5 adapter sequences.

  1. Lab Staff use either the 5nM dilutions or stock samples to measure concentration
  2. Sample is plated in triplicate at three different dilutions
  3. Plate is processed on QPCR machine
  4. Lab Staff reviews the raw data
  5. Jonathan loads the into the database

MiSeq pre-run sequencing

Since the NovaSeq generally requires us to pool multiple experiments we include a small MiSeq run. This run produces just enough data to ensure that the pool is probably balanced and that there are no barcode collisions.

Pooling

We generally pool without further dilution from the ‘5nM’ stocks. This means we generally put a different volume of each sample into a new tube and add TRIS to dilute to the final target molarity.

We plug the Kapa-derived molarties for the ‘5nM’ dilution into a prepared spreadsheet. We pick a nominal target molarity and volume and see what sample volumes the spreadsheet indicates.

Interacting with NGSC

Equipment Details

Experimental Techniques

Retrieving Data

Penn Links

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