Frequently Asked Questions

Below are some of the most commonly asked questions we receive from clients. If you have additional questions please feel free to contact us for more information.

Sequencing

What type of genome sequencing can you perform?

Our facility is equipped to do the following types of sequencing:

  • DNA & RNA Sequencing
  • Bacterial, Viral, and Targeted Human Sequencing
  • Exome Sequencing
  • Whole Genome Sequencing
  • Targeted Sequencing

What organisms can you sequencing?

We can do whole genome sequencing on:

  • Bacteria
  • Bacteriophages
  • Viruses
  • Fungi
  • Parasites
  • Yeast

We can perform targeted sequencing on:

  • Humans
  • Animals
  • Plants

Can you create custom gene panels?

Yes! We have the capability to target any gene within the human genome. We often work with researchers who are doing specific research and need to target certain genes. Our custom gene panel service is provided at no additional cost. Each targeted gene costs between $19-$25/gene.

What is "crowd sequencing" and how does work?

Many researchers have a small amount of samples that they want to analyze. However, with the price of consumables and facility overhead, sequencing a single sample is often cost prohibitive.

“Crowd sequencing” is the idea that we group multiple similar sequencing projects into a single run for various researchers in order to provide them the benefit of cheaper per sample pricing.

The way it works is that we provide an estimated price range that each researcher agrees to. The researcher send their samples in and we house them in our freezers at no cost. From there, we work to identify candidate sequencing runs that have extra capacity or we source enough researchers to create a new run within the estimated price range.

Since most facilities require a minimum amount of samples or are too expensive to do small sample runs, Crowd Sequencing is a great option for researchers who aren’t under a time crunch but are price sensitive. To learn more, get in touch with us.

How do you quantitate DNA concentration?

When we receive samples in the Lab, one of the first steps we perform is DNA quantitation to determine the sample DNA concentration.  We use Qubit 3.0 fluorometers and Qubit assays for this task.

The Qubit dsDNA BR (Broad Range) Assay Kit (https://bit.ly/2wb3wgr) is designed to measure double-stranded DNA concentration in the range 100 pg/uL – 1,000 ng/uL, or a mass range of 2 – 1,000 ng.  In practice, we generally use the BR assay kit to determine dsDNA concentration down to 1 ng/uL.

The Qubit dsDNA HS (High Sensitivity) Assay Kit (https://bit.ly/2fd765o) is designed to measure double-stranded DNA concentration in the range 10 pg/uL – 100 ng/uL, or a mass range of 0.2 – 100 ng.  In practice, we generally cut-over from the BR assay kit to the HS assay kit when dsDNA concentration is below 1 ng/uL.

qubit 3.0 fluorometer

HLA Typing

What are the minimum sample requirements for HLA typing runs?

We use the Illumina TruSight HLA v2 Sequencing Panel for HLA typing runs.  The minimum sample requirements for this assay are:

  • DNA mass:  2 µg
  • DNA concentration:  20 ng/µL
  • Sample volume:  100 µL

NOTE:  If necessary, we can work with lower sample volumes and/or lower DNA concentrations, but it requires some modifications to our library prep protocols. The absolute minimums are sample volume = 50 µL and DNA concentration = 10 ng/µL.  Please contact us if you have any questions about minimum sample volumes or DNA concentration.

What type of report is generated from an HLA typing run?

We use the Illumina TruSight HLA v2 Sequencing Panel for HLA typing runs. And we use the Illumina TruSight HLA Assign 2.1 software application to analyze HLA typing results.  Assign compares sample sequences against a standard library of sequences in the IMGT (International Immunogenetics Information System; www.imgt.org) HLA database.

An example of the type of output generated by Assign is shown below:

Sample names are shown highlighted bold in the first column.  HLA gene names and gene loci are shown highlighted bold in the first row.  The 11 HLA gene loci included in the TruSight HLA v2 Sequencing Panel are listed here.  

For each sample and gene locus, two rows are shown – one for each allele.  Assign typically displays three fields of resolution, i.e “00:00:00”. Complete fields indicate unambiguous typing results. A double-dash “–” indicates an ambiguous field in the typing results.  An “X” for the second allele indicates that no heterozygous positions were detected in the sequence. Bold text indicates a CWD (common and well-documented) allele. (Typically, there is a substantial number of CWD alleles).  The DRB genes and alleles form a complicated set of paralogues and pseudogenes. The blank fields under DRB genes reflect this complexity; a full discussion of these blank fields is beyond the scope of this note.

We include this type of HLA report with all HLA runs.  And we include Assign software documentation in case you need to drill down for more detailed analysis of the results.  

Links:

https://www.illumina.com/products/by-type/clinical-research-products/trusight-hla-v2.html

https://support.illumina.com/downloads/trusight-hla-analysis-software-assign-2-0.html

Bioinformatics

What is the file naming convention for FastQ files stored in Box?

When sequencing runs are finished, we automatically upload FastQ files to a clients Box.com account.  These files are typically found in the directory:

ClientName->Reports->ReportID->FastQ

The FastQ files in this directory adhere to the following naming convention:

ClientName_ProjectID_SampleID_SampleName_sometext_R1_sometext.fastq.gz

ClientName_ProjectID_SampleID_SampleName_sometext_R2_sometext.fastq.gz

 

ClientName = the name of the client’s organization

ProjectID = a SeqCenter internal project identifier (you can generally ignore this)

SampleID = a SeqCenter internal sample identifier (you can generally ignore this)

SampleName = this is the sample name supplied to the SeqCenter by the client; it typically corresponds to the sample vial name

sometext = a text string added by the sequencer (you can ignore this)

R1 = the forward read identifier

R2 = the reverse read identifier

fastq = identifies this as a file in FastQ format

gz = the files are compressed in gzip (“.gz”) format

In most, but not all, sequencing runs we use paired-end reads.  There is a forward read and a reverse read, which together form a single paired-end read.  The R1 and R2 monikers refer to these individual reads. The forward reads and reverse reads are stored in separate files.  Many bioinformatics applications expect these two files to be processed together so you can keep track of them with the R1 and R2 identifiers.  

General

What does your pricing include?

We’re unique in the industry in that we provide all-inclusive pricing. This pricing includes:

  • DNA Extraction
  • Sample Quality Control
  • Library Preparation
  • Genome Sequencing
  • Basic Bioinformatics (Sequencing Alignment & Variant Analysis)
  • Short term data management (secure file hosting for transferring outcome data to you)

For sample extraction, if you want to perform that yourself then we will remove that cost from the all-inclusive pricing. Our goal is to provide sequencing to our client at the lowest possible rate.

How long does it take to get genome sequencing performed?

End-to-end genome sequencing varies greatly based on the type of sample as well as the sequencing method. For example, bacterial whole genome sequencing typically takes 24 hours to run on the sequencer alone. However, there is pre and post sequencing time that is required so it can take anywhere from 3-7 days.

If you need a concrete estimate, get in touch with us and we can provide a turnaround time estimate.

How do I submit samples?

We have a comprehensive sample submission guideline available. Still need help? Feel free to shoot us an email at info@thesequencingcenter.com to ask your question or head over to our Contact Us page.

Do you collaborate with researchers on projects?

Absolutely. Our staff is invested into progressing research and have collaborated on many research projects. We often provide collaboration in the form of research, protocols, and data analysis from the genome sequencing we perform. For novel research fields, we can contribute to research papers via custom protocol creation, gene panel creation, gene targets, and bioinformatics analysis.

What Biosafety rating does your lab have?

Our facility has been certified as a BSL2 facility. Certain organisms that are typically handled in BSL3 labs can be accepted by our facility in specific scenarios. To see if we can handle your organism or contribute to your research, get in touch with us.