Illumina Whole Genome Sequencing

Illumina Whole Genome Sequencing Illumina Whole Exome Sequencing

The Illumina WGS pipeline is a common workhorse for many downstream applications and is our most popular service. Keeping scaling in mind, our packages are sized per sample and state the minimum total amount of sequencing data produced for each package. All Illumina WGS packages include library prep and sequencing at a price point made possible by multiplexing many samples from multiple orders on shared flowcells. All prepared libraries are indexed with IDT 10 bp UDI indices and later bioinformatically demultiplexed after sequencing.

Our WGS pipeline relies on the Illumina DNA Prep kit, which is flexible enough to accommodate all sizes of genomes. This system performs an unbiased, bead-based tagmentation to consistently produce inserts roughly 280bp in length, which is ideal for the 150bp paired end read output. Crucially, this library preparation kit also enables us to accept a wide range of sample concentrations (~1-500ng) and is tolerant of many sample impurities, with few exceptions. This flexibility allows us to consistently deliver high-quality prepared libraries for a wide range of sample types, though it does require starting dsDNA fragments longer than 400bp for successful preparation. For additional information about concentration requirements and impurities of note, see below.

Sample Requirements:

  • Total volume of 30 µL or more
  • Eluted into water or dilute Tris/TE buffer
  • Sample concentration greater than:
    • 10 ng/µL measured by Qubit
    • 20 ng/µL measured by Nanodrop
  • Fragment length of at least 400bp

What You Will Receive:

  • Two fastq files (2 x 151bp) for each sample
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Globus within 2 weeks

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
200 Mbp (1.33M Reads)
Example uses: Plasmids, viral dsDNA, bacterial genomes <5Mb		 $75.00
400 Mbp (2.67M Reads)
Example uses: Bacterial genomes >5Mb in size		 $95.00
650 Mbp (4.33M Reads)
Example uses: Bacterial populations, yeast genomes		 $105.00
1 Gbp (6.67M Reads)
Example uses: Large yeast genomes, simple metagenomes		 $120.00
2 Gbp (13.3M Reads)
Example uses: Diverse metagenomes, small algae		 $150.00
5 Gbp (33.3M Reads)
Example uses: Genomes <150Mbp		 $250.00
10 Gbp (66.7M Reads)
Example uses: Genomes <320Mbp		 $320.00
25 Gbp (167M Reads)
Example uses: Genomes <820Mbp		 $400.00
Mouse/Human 30x
A minimum of 100Gbp of 151bp PE Reads		 $550.00
Mouse/Human 60x
A minimum of 200Gbp of 151bp PE Reads		 $1,050.00
*Orders >48 Samples receive 5% discount

We offer convenient DNA extraction services using ZymoBIOMICS DNA extraction kits. This protocol can extract DNA from even tough-to-lyse cells, using a bead bashing tube for consistent mechanical lysis. We are able to accommodate all organisms up to BSL2+ and do not accept BSL3 or higher classified organisms for extraction services.

Samples requiring DNA extraction can be submitted as aspirated cell pellets shipped frozen on dry ice or as growth on sealed, nutrient agar plates shipped at ambient temperature. A minimum of 109 cells is recommended for all microbial DNA isolation submissions. We cannot culture samples; we encourage you to ensure samples are pure and that there is adequate growth before shipping.

Please note that while this kit is broadly applicable to many sample types, we cannot guarantee its efficacy for a given organism. More information about the ZymoBIOMICS kit can be found here.

We offer a selection of basic bioinformatic services to get you started with analyzing your data. These services are meant to be the first step into subsequent investigation and extensive validation studies. Our standard offerings include haploid variant calling and de novo short read assembly and annotation services. For more information on these services or custom services like reference-based scaffolding, please see our bioinformatics service pages.

Sample Format and Shipping

For shipments of less than 48 samples, we recommend sending individual microcentrifuge tubes, labeled with permanent marker to match the order and sealed with parafilm. DNA can be shipped at ambient temperature, though expedited shipping and dry ice can preserve fragment length and total material, which may be preferred for low concentration samples.

For orders over 48 samples, we also accept 96-well PCR plates sealed with a plate seal; we highly recommend shipping 96-well plates on dry ice to reduce the risk of well-to-well contamination.

More shipping information is listed at the end of the ordering process and on our FAQs page.

Submission Considerations

A few factors can hinder a successful Illumina DNA prep or result in less-than-ideal data, namely: low dsDNA concentration, incompatible sample types, and inhibiting impurities.

  • dsDNA concentration: The Illumina DNA prep is very successful in sequencing low input samples, but there are a few key factors to take into consideration:
    • Accurate measurement: We strongly recommend a dsDNA-specific, dye-based quantification method, such as Qubit or PicoGreen. These provide the most accurate measurement of concentration. Other absorbance-only methods, like Nanodrop, regularly overestimate concentrations (by orders of magnitude), as many artifacts commonly present from DNA extractions absorb at the same wavelength measured, such as all carbohydrates.
    • Preserving material: In general, Illumina WGS samples with Qubit concentrations above the requirements can be shipped at ambient temperature, as DNA is slow to degrade. However, we recommend dry ice or cool packs for lower concentration samples or heavily degraded samples to preserve sample integrity as much as possible.
    • Correlation to unique reads: The concentration requirements listed are well within a range that will provide ample amounts of unique reads, with few PCR duplicates. The prevalence of PCR duplicate reads increases as concentration decreases.
    • Extremely low concentrations: We have some options available for processing very low input samples, though we ask that you contact us before shipping samples to discuss the options. Special handling fees may apply. However, extremely low concentrations (< 1 ng/µL) are unlikely to succeed library prep.
  • Incompatible sample type: This pipeline is intended for whole genome samples, which means that other sample types may not be processed as expected. Preparing a sequencing library and submission through our dedicated Illumina Full Flowcell pipeline may be an option.
    • Minimum fragment length of 400bp: For tagmentation, the enzymes are spaced on the magnetic beads to force a narrow range of insert sizes but also impose a minimum starting fragment length required.
    • Loss of linear ends: Linear fragments will receive little to no coverage of the terminal 50bp on any fragment. For random fragmentation of most genomes, this is not an issue but can be for linearly fragmented genomes or PCR products.
    • Implicit fragmentation: All starting material will be fragmented, and any constructs will be broken apart, separating ends.
    • Interference from other barcodes, primers, or sequencing adapters: Pre-prepared sequencing libraries are NOT compatible with this pipeline and may result in unsuccessful preparation or demultiplexing errors resulting in missing or contaminated data.
    • Other unique considerations: In some very rare instances, highly specific base modifications may not be compatible with the Illumina DNA Prep kit. (Some phage-specific examples have been reviewed.)
  • Potential inhibitors: Though the Illumina DNA prep is very robust, there are a few key impurities (sometimes residual from DNA isolation) that can interfere with the enzymes or magnetic beads used in library preparation. While this is not an extensive list, the most common inhibitors that we encounter are listed below.

Potential Inhibitors

Inhibitor  Source  Action  Solution 
EDTA  Commonly used in elution buffers  Can inhibit PCR by depleting Mg2+ ions which are essential cofactors for proper DNA polymerase activity  Dilute EDTA to 10 mM prior to resuspension of DNA
Phenol  Commonly found in phenol chloroform-based DNA extraction kits  Can denature polymerases  Additional purification or clean-up  
Ethanol   Commonly used during DNA purification  Strong inhibitor of polymerase activity  Allow ethanol to completely evaporate before final elution or resuspension
Ionic detergents (SDS, sarkosyl, sodium deoxycholate, CTAB)  Commonly used in extraction kits to disrupt cell membranes and denature proteins  Strong inhibitor of polymerase activity  Additional purification or clean-up 
Iron and metallic DNA polymerase inhibitors   Growth media, environment  Interfere with the action of the tagmentation beads or DNA polymerase  Column-based purification

Additional Resources

Illumina Whole Exome Sequencing

Providing an economical alternative to WGS of the human genome, our whole exome sequencing approach focuses on sequencing the protein-coding regions. This approach utilizes a collection of panels to cover genes that are linked to phenotypes useful for biological, medical, and public health applications.

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Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

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Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

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