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NGS FAQ - CGP Illumina

  1. Why do I need to submit my sample at 10 nM?
    A: Illumina recommends a concentration of 10 nM for long-term storage of DNA libraries.  We store all original samples at the Core indefinitely.  The libraries are diluted to 2 nM working concentration, for denaturing with 2 N NaOH to a final concentration of 1 nM.  In our experience the optimal denaturing is achieved following this protocol.
  2. My library is 6 ng/µL measured on the Nanodrop.  Is this concentrated enough?  How do I convert to nM? 
    A:   A target concentration of 5- 10 ng/µL is usually adequate for us to work with.  However, the nM concentration of your sample is dependent on the fragment size.  The average molecular weight of 1 nucleotide pair is 660 g/mol.  To convert ng/µL to nM:

    For example, 1.9 ng/µL library of 290 bp fragment size is ~10 nM.
  3. I submitted my libraries and the sample submission form- what’s next?  Will I be informed of the QC results?
    A:   We process samples in the order that we receive them.  First, the sample will be run on the bioananlyzer at a concentration of 500 pg/µL.  There are 11 wells per Agilent bioanalyzer High Sensitivity chip and we wait to run a full chip to maximize reagent use.  If the libraries look good on the bioanalyzer (defined peak of relatively narrow region and no adapter contamination), then we proceed to quantify the samples with qPCR, using the KAPA Biosystems’ primers specific to Illumina adapters. 
  4. Due to the volume of samples we process, we do not generally inform users of the QC results, unless we are concerned about quality of the samples, or they fail QC. 
  5. If samples fail QC, you will be informed promptly.  Please join our listserv newsletter to receive updates on when samples will run. 
  6. When will my sample run on the HiSeq? 
    A:   Due to the unpredictable sample number received and the quality of the samples we receive, we cannot guarantee when a given sample will run.  The wait time depends on what type of run you choose and the demand for that run.  Since operation of our sequencing service, the average wait time is approximately 3-5 weeks from receipt of sample to start of the sequencing run.
  7. Why do I have to wait for a flow cell to “fill up”  before my samples are run?
    A:   The Illumina flow cell is a sophisticated glass slide with 7 lanes on which clustering and sequencing take place.  The expense of reagents and the workflow of the HiSeq 2000 prohibits running flow cells with empty lanes (unless you are willing to pay for empty lanes).  For this reason, we wait until we have 7 lanes of samples to start a sequencing run.  We expect to upgrade to the HiSeq 2500 in the spring of 2013, which will allow us to run flow cells with 2 lanes, thus decreasing wait time for the user.
  8. Do you spike the libraries with a PhiX control library?
    A:   No, we do not spike samples with PhiX unless requested by the user.  For optimum QC of sequencing procedures, we dedicate one lane on every flow cell to a PhiX control.  If your library is known to be of low diversity, you may choose to spike PhiX to maximize cluster differentiation and sequence quality.  For example, you may specify on the sample submission form that you want 25% PhiX and we will combine PhiX accordingly before clustering on the flow cell.  Please contact us if your library has low diversity.  Low diversity can occur, for example, when sequencing bacterial ribosomal DNA to identify species composition in soil samples. 
  9. What library concentration gives the optimal cluster numbers on the flow cell?
    A:   The clustering efficiency depends on library quality and insert size, and is different for every sample.  In general, over-clustering will reduce the sequence data quality, while under-clustering will result in reduced yield.  Therefore, titration of the library would be ideal to determine the optimum cluster density of any given sample.  However, this is generally cost prohibitive.  Based on our empirical experience with the performance of the HiSeq, we routinely load 9 pM of sample library for clustering on the flow cell, and 12 pM of PhIX.  The sample concentration may be lowered if over-clustering occurs and the sequence run must be repeated.
  10. What is an Oscar account?  How do I set up and Oscar account?
    A: “OSCAR” is the name of Brown’s computer cluster for high performance computing maintained by the Center for Computation and Visualization on campus.  The cluster has several hundred multi-core nodes which are used for sequence analysis, among other shared jobs.  After sequencing is completed, the data is sent from the local server at the HiSeq to OSCAR.  The Illumina sequence data is sent to OSCAR from our local server at the HiSeq.  All users of the Brown Illumina sequence service will need an Oscar account to work with their data.  To request an account, choose the “Bioinformatics” link on the left hand side of our Illumina sequencing page.  Then choose “CCV HP cluster Support” on the lower left and follow the instructions to request an OSCAR cluster account. 
  11. I have 14 Ilumina prepared multiplexed libraries.  Should I pool them before submitting to the Core?
    A:   No, you do not need to pool your original samples before submitting to the Core. 
  12. For the most accurate representation of each multiplexed sample in a pool, we prefer to complete QC on each individual samples.  This way we can exclude bad libraries and match the concentration in the final pool.  Unless otherwise instructed on the sample submission form, we will pool equal molar amounts of each sample.  We store the original sample, the 2 nM dilution, and the final pooled sample indefinitely (or until freezer space runs out).
  13. I ran a sample over a year ago.  Can I re-sequence it at the Core or retrieve the original sample?
    A:   Certainly.  We store all original samples (including samples that fail QC), the 2 nM dilutions, and the pooled samples indefinitely at the Core Facility for your convenience, at no extra cost.  You may request a sample to be re-sequenced here, or retrieve the original sample.
  14. How do I get in the queue for the next 50 bp SR run?
    A:   By submitting your libraries with an accompanying sample submission form (please submit both paper and electronic), you are automatically entered in the queue for QC.  After your samples pass QC, they will be sequenced on the next available run.  We track submission dates and times to facilitate the “first come, first serve” model.  You don’t need to do anything else to “hold” a place in a future run.
  15. Does the Brown Genomics core do library prep?
    A:   At this time we do not offer library prep as part of our services.  However, URI is offering library prep to the Brown community.
  16. I received the First base Report for my sample’s sequencing run.  How do I interpret the report?  Which lane(s) contain my sample?
    The first base report is our first indication on the HiSeq of the sequencing quality of a given run, and provides a quality control step to decide whether to proceed with the run.  Should anything go drastically wrong, the first base report will allow us to identify the problem before committing to the run and using up the expensive sequencing reagents.  The first base report is saved in word document format and sent to each of the users on the run.  The metrics supplied are: cluster density, intensities of each of the 4 bases, and a focus score for each base.  The cluster density is sample dependent, but should be between 250-400 k/mm2.  This is a first approximation of the cluster number, and will be more accurately reported as the run proceeds.  However, the first base report is a useful rough estimation.  The intensities of the bases also vary, as the bases A and C are read by one laser and G and T are read by another.  You can expect intensities around 20K for A/C and 10-20K for G/T.  A focus score of 100% is perfect, but as low as 70% will still yield quality sequence data.
    The first base report does not contain lane assignments.  We record this information in a Google document called “HiSeq 2000 run log”.  We share this document with each user on a sequencing run, at the start of the run.  You can expect to receive both of these documents within 24 hours of a run started.