GENERAL
Where are you located?
University of Washington Foege Building South, 3720 15th Ave NE, Seattle, WA 98195
Do you archive submitted samples? Do you return samples?
Yes. We keep samples in the -80 for deep storage for 5 years. We can return samples upon request if they are within the timeframe.
Do you provide Bioinformatics help?
Depends on the task. Please send a request to nwgc@uw.edu.
How do I acknowledge your services?
Please support us by acknowledging our services in your publications. You can thank the Northwest Genomics Center at the University of Washington. Acknowledgments like this are a big support for future NIH instrumentation grant applications. Thanks in advance!
Do you ask for co-authorships?
We ask you to mention our services in the acknowledgments of your publications.
Where can I find a tutorial on Short-Read NGS sequencing? Long-Read?
For Short-Read sequencing, check out: Next Generation Sequencing – A Step-By-Step Guide to DNA Sequencing
Watch the videos of the different types of short-read sequencing chemistries and how they work here: https://winterdev.cc/nwgc-dev/services/structural-genomics/short-read/
For Long-Read sequencing, check out: Long Read Sequencing Comparisons
FULL PIPELINE
Sample Submission
What types of samples are accepted?
We accept DNA and RNA samples that are plated in our designated barcoded plates.
What are the DNA or RNA requirements for submission?
Requirements for submission vary depending on the scope of a project. Our general guidelines for Short-Read Sequencing are:
WGS: 2ug = 40ul of 50ng/ul in TE
Exome: 2ug = 40ul of 50ng/ul in TE
RNA: 1.5ug = 30ul of 50ng/ul in Nuclease Free Water, RIN >7
When submitting a manifests for multiple plates, can I submit one spreadsheet, but put the different plates on separate tabs?
No, please submit one manifest spreadsheet per DNA plate.
Can I use subjects first and last names as sample IDs?
No, do not use subject names. Please do not send any information that links to subject identity.
Can I alter the submission manifest?
You may add columns, but do not remove columns or change the names of any of the columns.
There are hidden rows in the manifest, may I remove them?
No, those are hidden rows that are needed for entering the manifest into our LIMS.
May I send my samples at room temperature?
Since we do not accept samples in screw-top lids, please do not send samples at room temperature. Samples that are packed frozen and that stay frozen during delivery are much less likely to contaminate each other.
I am in the Seattle area. May I pick up my plate and bring it back when I’ve aliquoted out my DNA?
Yes, please feel free to drop by the lab. We can send instructions for how to reach us.
Which DNA isolation protocols do you recommend for Illumina sequencing?
We recommend DNA isolations with spin-column kits/protocols that should include an RNase digestion step.
Can you run samples with less than the recommended input material?
This will be determined by the Project Manager.
Can I submit samples of lower integrity than recommended?
This will need to be discussed prior to sample delivery with the Project Manager.
How should I purify my samples? How should I remove DNA or RNA contamination?
We suggest bead based cleanups or spin-column cleanups. DNA needs to be RNA-free (use RNase) and RNA needs to be DNA-free (use DNase). Always check for chemical contamination using spectrophotometry (Nanodrop) and concentrations using fluorometry (Qubit/Quant-It). Nanodrop: For RNA the 260/230nm ratio should be >1.5 and the 260/280nm ratio 1.8-2.1; For DNA the 260/230nm ratio should be >2 and the 260/280nm ratio 1.8-2.0. There are a variety of kits for purifying DNA and RNA, please check to see which is best for your experiment and follow provided protocols.
Do you offer DNA isolations and RNA isolations as a service?
Unfortunately, we do not offer isolation at this time.
What type of library services are available through the NWGC?
Please check the options under ‘Services’ menu.
QC
What happens to samples that fail QC?
Samples that fail QC are not eligible to proceed into the sequencing pipeline. A single sample may be excluded from the project or in cases where the failing sample is critical to the analysis (i.e. the proband in a trio) the entire family will be excluded.
I’ve received notification that one or more of my samples has failed QC?
If a clerical error on the manifest has caused the sample to fail the sex check, you may notify the NWGC of the need to correct a manifest. If a sample has failed due to a sex conflict or due to insufficient concentration, you may submit a replacement sample by filling out a new manifest describing the replacement sample. Submission of replacement samples will delay the release of data for your project.
Sequencing
What is the turnaround time for sequencing?
Turnaround time greatly depends on the size and scope of a project and what is currently in our queue. However, on average it takes approximately 8-12 weeks after all samples in the project pass QC for data to be released. Projects with a large number of samples may require extra time for sequencing before all samples are finished.
What read numbers/yields can I expect from the different sequencing platforms?
| Sequencing Platform | Cycles (bp) | Number of Reads *paired-end |
| Illumina® MiSeq | 50, 300, 500 | ~30 million for V2 ~50 million for V3 |
| Illumina® NextSeq 500 MO | 75, 150, 300 | ~260 million |
| Illumina® NextSeq 500 HO | 75, 150, 300 | ~400 million |
| Illumina® NovaSeq 6000 SP | 100, 200, 300 | ~1.6 billion |
| Illumina® NovaSeq 6000 S1 | 100, 200, 300 | ~3.2 billion |
| Illumina® NovaSeq 6000 S2 | 100, 200, 300 | ~8.2 billion |
| Illumina® NovaSeq 6000 S4 | 35, 200, 300 | ~20 billion |
| Illumina® NovaSeq X Plus 1.5B | 100, 200, 300 | ~3 billion |
| Illumina® NovaSeq X Plus 10B | 100, 200, 300 | ~20 billion |
| Illumina® NovaSeq X Plus 25B | 300 | ~50 billion |
| Element AVITI™ Cloudbreak Freestyle Low Output | 300 | ~250 million |
| Element AVITI™ Cloudbreak Freestyle Mid Output | 150, 300, 600 | ~500 million *600 cycle kit ~100 million |
| Element AVITI™ Cloudbreak Freestyle High Output | 150, 300, 600 | ~1 billion *600 cycle kit ~300 million |
| Long-Read Sequencing Platform | Flowcell Minimum | Output (Gb) |
| PacBio Revio™ | 1 SMRTcell (Run Time: 12, 24, or 30hrs) |
~60-90 |
| Oxford Nanopore Technologies® | 1 flowcell | ~50-150 |
Data Delivery
How will I know when my data are available?
You will receive an email notifying you of data release.
What data do I get back from the NWGC?
Please see https://winterdev.cc/nwgc-dev/tech-platforms/bioinformatics/pipelines/
Can the NWGC provide FASTQ files in addition to bam files?
You can generate FASTQ files from the BAMs received in data release. Instructions.
Do you archive the sequencing data?
Yes. Depending on the type of data. Our default is to keep data on tape for 4 months. Short Read bams are kept for 7 years on tape and VCFs 1 year. Long Read bams are kept for 3 years on tape.
ALREADY PREPARED LIBRARIES
General
What types of samples are accepted?
We accept already constructed libraries that are ready to be sequenced.
What are the volume and concentration requirements for sample submission? The minimum molarity for any library submission is 5nM, but the minimum volume depends on the flowcell type. Please see the below table:
| Sequencing Platform | Minimum Molarity | Minimum Volume |
| Illumina® NovaSeq S4 | 5nM | 130uL |
| Illumina® NovaSeq S2 | 5nM | 55uL |
| Illumina® NovaSeq SP/S1 | 5nM | 35uL |
| Illumina® NovaSeq X Plus 1.5B | 5nM | 20uL |
| Illumina® NovaSeq X Plus 10B | 5nM | 60uL |
| Illumina® NovaSeq X Plus 25B | 5nM | 100uL |
| Illumina® NextSeq 500 MO or HO | 5nM | 10uL |
| Illumina® MiSeq | 4nM | 20uL |
| Element AVITI™ Cloudbreak Freestyle | 5nM | 20uL |
| Long-Read Sequencing Platform | Minimum Concentration | Minimum order |
| PacBio Revio™ | 20-60ng/ul in 28ul (concentration varies based on insert size) | SMRT™ Cell |
| Oxford Nanopore Technologies® | 2-10ug (concentration varies based on prep type and coverage needed) | Flow cell |
May I send my samples at room temperature?
Please do not send samples at room temperature, unless the sample is already at room temperature and is being dropped off in person. Samples that are packed frozen and stay frozen during delivery are much less likely to contaminate each other.
I am in the Seattle area. May I drop off my sample directly to your lab?
Yes! Once the quote and sample submission form are complete and you are ready to drop off your sample, please contact us for directions and to schedule a drop off.
Can I submit custom primers with my samples?
Yes, if necessary. The amount will vary depending on the platform used, but typically 20uL of 100uM primer is sufficient.
I do not need a lot of data, can I order part of a lane?
No, we do not sell partial lanes. We recommend either choosing another platform that generates fewer reads or waiting until you have more samples to submit.
What is the turnaround time for sequencing?
Turnaround time greatly depends on the number of samples submitted and the availability of the sequencers. The typical turnaround time is 2-4 weeks for sequencing only projects.
Do you discard samples after a certain amount of time?
No. Currently, we do not have a contingency plan for discarding submitted samples for sequencing only projects.
Can I submit samples for QC only?
Yes! Please email nwgc@uw.edu for a quote and then email nwgcseq@uw.edu for the following steps.
How should I submit the barcode sequence information? In which direction will they be sequenced?
On the Sample Submission sheet, please submit all barcodes as i7 and i5 forward, with their designated sample ids. This can also be sent in a separate excel doc if there are many. For 10X indices, please provide the SI-XX-XX name and the sequences for each, including all 4 sets of barcodes for 10X ATAC preps. As for direction of sequencing, depending on sequencer, the second index (called i5 index) will be read in different orientations, but our LIMS system has pipelines built out to demultiplex the barcode in the correct orientation based on the sequencer.
How do I pool sequencing libraries? Can you pool them for me?
If your libraries are already prepped, QC’d, and quantified using qPCR, you can balance them depending on how much of each library you want in the pool/how many reads/coverage you need. If you want even coverage across all samples, you can pool them equally based on their final nM. If samples are not QC’d or you are not sure on the pooling, we can perform the QC here on each library and pool based on coverage needed per sample. We will also perform QC on all incoming pools or individual samples before proceeding with any sequencing. All QC is reported to the user prior to sequencing.
Can I submit a library I made?
Yes, please see https://winterdev.cc/nwgc-dev/services/already-prepped-libraries/getting-started/
What read numbers/yields can I expect from the different sequencing platforms?
| Sequencing Platform | Cycles (bp) | Number of Reads *paired-end |
| Illumina® MiSeq | 50, 300, 500 | ~30 million for V2 ~50 million for V3 |
| Illumina® NextSeq 500 MO | 75, 150, 300 | ~260 million |
| Illumina® NextSeq 500 HO | 75, 150, 300 | ~400 million |
| Illumina® NovaSeq 6000 SP | 100, 200, 300 | ~1.6 billion |
| Illumina® NovaSeq 6000 S1 | 100, 200, 300 | ~3.2 billion |
| Illumina® NovaSeq 6000 S2 | 100, 200, 300 | ~8.2 billion |
| Illumina® NovaSeq 6000 S4 | 35, 200, 300 | ~20 billion |
| Illumina® NovaSeq X Plus 1.5B | 100, 200, 300 | ~3 billion |
| Illumina® NovaSeq X Plus 10B | 100, 200, 300 | ~20 billion |
| Illumina® NovaSeq X Plus 25B | 300 | ~50 billion |
| Element AVITI™ Cloudbreak Freestyle Low Output | 300 | ~250 million |
| Element AVITI™ Cloudbreak Freestyle Mid Output | 150, 300, 600 | ~500 million *600 cycle kit ~100 million |
| Element AVITI™ Cloudbreak Freestyle High Output | 150, 300, 600 | ~1 billion *600 cycle kit ~300 million |
| Long-Read Sequencing Platform | Flowcell Minimum | Output (Gb) |
| PacBio Revio™ | 1 SMRTcell (Run Time: 12, 24, or 30hrs) |
~60-90 |
| Oxford Nanopore Technologies® | 1 flowcell | ~50-150 |
QC
Will my samples go through quality control before they are sequenced?
Yes! All samples that are submitted as sequencing only projects require QC prior to sequencing. Each sample will be run on the Qubit and the Bioanalyzer. The QC will be reported back to the user before any sequencing begins to ensure the libraries are what is expected.
What happens if my sample fails QC?
Samples that fail QC are reported back to the user and they can reprep their libraries.
My libraries show peaks larger than expected. Can I still sequence these PCR-bubbles?
This PCR-bubble is an artifact of annealing products and the resulting libraries are perfectly sequence-able. However, the quantification via the qubit will not be accurate. Bead clean ups will not typically get rid of the bubble either. Going forward, the protocol used to prep the library should be optimized to use a lower number of PCR cycles.
Data Delivery
Do you demultiplex the sequencing data?
Yes, the demultiplexing is including in the price of sequencing as long as the barcodes are provided and there are no custom primers/pipelines needed. FASTQs are the deliverable.
How will I know when my data are available?
You will receive an email from nwgcseq@uw.edu notifying you data is available for download via Globus.
What data do I get back from the NWGC?
For Sequencing Only Projects, raw run data is converted to FASTQ format and placed on our server for you to download. If you require any raw data or run files, there is an additional bioinformatics fee based on an hourly rate.
When will I receive my data?
Our typical turnaround time is 2-4 weeks from submission to data being ready for download. This time may be shorter or longer depending on our current workload.
My FASTQ file contains some “N”s. Is there a problem with my data?
An “N” means no call was made for that base. Reads at the beginning and end of sequencing data files originate from the edges of the flowcells, where imaging is more difficult, thus these reads show below average quality. It’s suggested to verify the quality of the dataset using programs like FASTQC.
Globus
Do you have a Globus server endpoint?
UW has the lowest bracket of Globus service so there are limitations. Please understand that Windows Server is not an official supporter of the platform for the Globus Connect Personal (GCP) software. The Windows versions officially supported for GCP are listed in our doc here. Some users do report success using GCP on Windows Server.
Can I upload my Globus data right to AWS?
Please consider the above questions answer and troubleshoot using the link provided via Globus found here.
How long will my data be available on Globus?
Once emailed that your data is ready on Globus, you will have 120 days to access it before it is deleted indefinitely.
GENOTYPING
General
How long will it take to receive my results?
Sample timeline is highly dependent on project sample size and QC resolution turnaround time. Typically this ranges from 7-28 days. If there are QC fails and replacement samples are being sent, this will delay release dates. Our project managers will do our best to communicate estimated timelines with you.
QC
What happens to samples that fail QC?
Samples that fail QC are not eligible to proceed. A single sample may be excluded from the project or in cases where the failing sample is critical to the analysis (i.e. the proband in a trio) the entire family will be excluded.
What if I’ve received notification that one or more of my samples has failed QC?
If a clerical error on the manifest has caused the sample to fail the sex check, you may notify the NWGC of the need to correct a manifest.
If a sample has failed due to a sex conflict or due to insufficient concentration, you may submit a replacement sample by filling out a new manifest and describing the replacement sample. New barcoded plasticware will be sent to you for your resubmission. Submission of replacement samples will delay the start of your project.
Why did my sample fail on the genotyping/methylation assay?
Each Illumina Infinium Array kit has a typical call rate cutoff that varies based on the chip used. (generally ~98%). The Illumina data analysis software calculates and reports a detection p-value, which represents the confidence that a given transcript is expressed above the background defined by negative and positive control probes, providing a good overall QC indicator. An unusually low number of detected transcripts could result from a number of causes such as high background on the array, low signal, poor stringency or poor sample quality. This sample may not have had a similar number of transcripts detected compared to all other samples on the BeadChip, resulting in a call rate below the assay threshold.
Globus
In what format will I receive my results?
Our project managers will work with you to generate a custom final report for your samples. The file type for your results will depend on the type of genotyping assay technology selected. Raw data files (idat) may also be sent upon request.
LONG READ SEQUENCING
What type of samples are recommended for the isolation of HMW-DNA?
The highest quality DNA can be isolated from cell culture and fresh blood.
10X GENOMICS – SINGLE CELL
Search the 10X Genomics Q&A page HERE.
Check out: https://www.10xgenomics.com/blog/faqs-about-single-cell-sample-preparation-covering-the-basics for 10X Genomics FAQs about Single Cell Sample Prep