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Sample Requirements, Submission, & Data


Library Design Requirements
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If you are new to Deep Sequencing, see the Resources section for basic information and helpful links.
The diagram below shows the basic structure of an Illumina library and the terms used on our submission ticket to describe its parts.

Diagram of Illumina library structure showing the P5 linker, i5 index, Read1 adapter and Read1/i5 primer site, inline barcode, insert, Read2 adapter and Read 2/i7 primer site, i7 index, P7 linker. The Core needs to know if there are barcodes, non-random, or homogeneous sequences in the inline and insert section.

Please fill out the service ticket(s) as completely and accurately as you can.

**Single and Dual Multiplexing are distinct from Single and Paired-End reads. Single multiplexed libraries have an index (called i7) at the P7 end, while dual multiplexed libraries have indexes at BOTH the P7 and P5 ends (i7 and i5). Each index that is placed in an adapter requires a separate read that is distinct from any insert reads (see diagram above), so Paired-End runs can have Single Indexing, and Single-Read runs can use Dual Indexing. For more details on multiplexing, download our Indexing and Barcoding document.

For those preparing their own libraries, we reserve the choice to hold back a sample which fails QC and contact you to discuss options. The use of a carrier agent during the final stages of workup is discouraged, as these can interfere with cluster formation and reduce the number of reads generated during sequencing.

**If you have added a spike-in control to your sequencing library, please be sure to specify that information on the ticket. Controls for normalization such as "Active Motif" or other antibody spike-ins that are not removed often have poor base-balance/repetitive motifs, and can badly overseed on a flowcell.

If you are doing a custom design to include adapters, your own index scheme, or custom sequencing primers, you must submit the results of your Topo cloning validation before we can run your samples. These sequences must span the P5 and P7 attachment sequences on either end of the library construct. If you do not agree to submit the sequence for verification, then you will be run with only the primers you request and at your own risk.

Downloadable Forms and Documents
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HiSeq/MiSeq Analysis Order Form ( Nov 2020 ) ( fillable pdf )

External (non-UMass) customer mandatory information form ( Intake Form )

Supplemental Sheet for Index List ( Sep 2021 )

Library Construction Order Form ( Apr 2020 ) ( fillable pdf )

Sample Submission Instructions ( click here for submission instructions )

DSCL workflow diagram ( workflow pdf )

Ticket Example With Tips ( example sheet )

Sample Submission Process
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Libraries should be submitted at 10nM concentration in 25μl of ultrapure water or EB. If your library doesn't have that high a concentration, don't panic! The Core will accept samples with concentrations as low as 2nM, depending on library quality at QC. Libraries below 2nM in concentration will only be run "at-your-own-risk", and outcome will not be guaranteed by the Core.

Please use 1.5mL tubes, 0.5-0.65mL tubes, or PCR strips that are labeled on the top of the tube. Do not submit single PCR tubes, and avoid long labeling strips that would prevent the tube from fitting in a centrifuge. In the Illumina pipeline, sample names are limited to 20 characters, which can include alphanumerics, "-", or "_".

Please note that a sample submitted for sequencing is entered in the workflow and queue, which engages time and services.  If you want the QC analysis only for your own information and are not certain you wish to move forward, please use the MBCL Fragment Analyzer or other QC methods (e.g. Topo Cloning, Gel Purification).

Libraries sent for sequencing should have one completed ticket PER LANE, and an Index List Form if applicable. Material sent for library building should also include a Library Prep Ticket. Electronic files are always welcome, especially for long index lists. If you are an internal UMass customer, the Core will accept an email from your PI authorizing charges in lieu of a signature. External customers must also complete a Customer Intake Form.
Typically we suggest that you send indexed libraries separately and let us do the mixing by molarity based on our internal QC. This helps with any potential troubleshooting if some samples in the mix underperform. However, if you prefer to premix your samples or choose a different mixing method (such as by volume), you are welcome to.

NOTE: It is VERY IMPORTANT that we receive the correct information about the primers and adapters used to build your library! Not all library types will run on all instruments or can be read in both directions.

If the kit or method you used for sample preparation does not work with the standard Illumina primer mix, please check off 'custom adapters' on the ticket and make arrangements for the Core to be provided with the custom sequencing primer.

If you are using the Illumina-style indexes located in the adapters, you must request a multiplex read step to detect your indexes. If you have used non-random sequence such as inline barcodes, linkers, spike-in controls, etc., please inform us by checking off the relevant section on the ticket.

Drop-off locations:
Samples can be left in the labeled fridges at these locations
for 11:00 AM pickup each business day:
Biotech2 outside Suite 207
LRB 6th floor mailroom
AS8-2016
We are also available by appointment if you need to visit with us.
Our shipping address for off-campus customers is
Rose-Gordon Bldg. Rm. 141, 222 Maple Ave, Shrewsbury, MA, 01545.

If you are shipping your sample(s), please do not ship for a Friday delivery! We will not be able to ensure they arrive in time to be properly stored before the weekend.

Sequencing Process
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The Core Lab staff will QC your submitted library and enter it in the sequencing queue. The Core will email you a copy of the QC results upon request. If you then choose not to move forward with sequencing, the Core will bill for the QC and processing to recoup costs.

Samples will be processed on a first-come, first-served basis. For runs involving multi-lane flowcells, this may include wait time until the run type is full. See our FAQ section for frequently asked questions about queue times.

You will receive an email notification when your data is ready. Instructions on how to retrieve your data will be included in the email.

Data Transfer
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The amount of data generated from a high-throughput run can be extremely large. We recommend eligible users get an account on the Green High Performance Computing Cluster for data delivery. See the Research Computing WIKI for more information. To get an account, contact the UMassMed Help Desk at 856 - 8643, email them at hpcc-support@umassmed.edu, or request access through this HPC form.

Alternatives for non-UMass customers are to have the data uploaded to an outside server (using SFTP) or transferred to an external drive meeting our requirements and shipped overnight.

  • Our current GHPCC data delivery system:
    (note that this changes as resources improve/upgrade, so stay tuned)

    • Your lab will have a directory at /project/umw_deepseq/FOR_DELIVERY/
    • You will get an email notification when your data files are ready.
    • If you require anything besides the fastq files, you must let us know at the time you submit your sample.
    • Data files will be deleted 5 business days after notification; please backup/copy/archive your data in your own workspace.
    • The DSCL does not have the resources to archive or recover data. Please ensure that you safely back your data up!

Data Analysis
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Please see the DeepSeq Data section for information about data analysis. If you would like to consult with an applications specialist, please email us to get on the schedule.

IF THERE IS A PROBLEM WITH YOUR RUN OR QUESTIONS ABOUT YOUR DATA, please let us know as soon as possible. The run metrics and machine files cannot be held for long because they are extremely large. The sooner we know there is a problem, the more likely we'll be able to help easily. In order to help sort out a problem we may ask a lot of questions and details about your sample and your analysis methods. This information is required in order for us to engage the tech support systems provided by our vendors.

Please note that if the control lane on the run failed in any way, the entire flow cell is rerun. If your data is delivered to you, then the controls all passed spec., which means that the instrument, reagents, and chemistry functioned properly. That leaves us with investigating other issues including sample design, base balance, sizing, indexing, degraded DNA, as well as computational and analysis issues with the pipeline, etc. We'll do whatever we can to get things working, but the more information we have, the better. You can find a troubleshooting guide for libraries here which is updated as new information is gathered.

Pricing and Payment Policy
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Please email DeepSequencingCoreLabs@umassmed.edu to get a quote for your desired services.

Processing and analyzing a sample requires time and reagents. Payment for these services is the responsibility of the user submitting the sample and should be rendered in a timely fashion. In the event of a reagent or equipment failure, the samples will be rerun at the next possible opportunity at no additional charge.

Clients withdrawing samples prior to the analysis run will be charged a fee to recover QC assay costs. For the return of archived post-analysis samples, clients will be charged a delivery fee per sample.

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