Package size recommendations are dependent on the customer’s project needs, sequencing platform, and genome size. On our service pages, we list example uses for each package size to give an idea of what package sizes are generally appropriate for various organisms.
Below are general calculations of package size for whole genome sequencing to serve as a guide. You need to know the coverage you are aiming for and an estimate of the expected genome size. As a general starting point, we recommend 30-50x coverage of your genome for variant detection and 100x combined coverage for assemblies.
Genome size (bp) × Coverage = Total minimum amount of sequencing required
Ex: 10 Mbp Genome × 30x coverage = 300 Mbp. (For Illumina WGS, a 400Mbp package would guarantee this amount of output.)
For transcriptome (RNA) sequencing, the data of interest are transcript counts, as opposed to coverage for whole genome sequencing. We strongly encourage customers to refer to comparable studies for starting estimates. Please see our RNA sequencing services for additional details.
For our dedicated flowcell offerings, flowcell choice will be dictated by how many output reads you wish to obtain, with considerations for inefficiencies (such as clustering for Illumina and ZMW to HiFi conversion rates for PacBio) and an optional PhiX spike-in for low diversity Illumina libraries. PhiX spike-ins decrease the total number of reads dedicated to your library. For example, a 25% PhiX spike in would mean that 75% of the reads will correspond to your library.
Coverage recommendations are often specific to the type of sequencing and the intended bioinformatic analysis.
As a general guideline for estimating single, clonal genome coverage, we recommend 30-50x coverage for variant detection and 100x combined coverage for assemblies, but certain study designs and analyses may necessitate even greater coverage. Please see “What size package should I order?” for a more detailed description of how coverage and package size are related.
Please refer to the individual service pages for service-specific requirements. All concentrations listed are minimums, and we do not generally recommend diluting samples.
We also recommend fluorophore dye-based quantification methods (such as Qubit or PicoGreen) for the most accurate dsDNA and RNA measurements, as purely spectrophotometric measurements, such as Nanodrop, tend to over-report material.
We ask customers to assess if their samples meet their own quality standards (keeping downstream analysis in mind), in addition to our requirements. We will attempt to sequence all samples we receive, as they are received.
SeqCenter will process all samples submitted, as they are. We strongly recommend that customers ensure their samples meet SeqCenter’s requirements for processing, as well as any additional or stricter requirements they might have for their projects. At a minimum, this should include quantification, preferably with a fluorometric method for the most accurate measurement. For RNA and any long read sequencing, fragment integrity or length analysis is also highly encouraged. Please see individual service’s page for specific requirements and recommendations.
Nanodrop and other non-dye DNA quantification measurements are acceptable, though we ask that you still meet at least the minimum concentration requirements for the appropriate service.
Non-dye-based methods tend to overestimate the concentration of DNA, often producing readings as much as 10 times higher than fluorometric measurements, as all other carbohydrates and organic compounds will also absorb in this range. Aiming for at least double the required amount can be a strategy to combat this, and measurements over 300 ng/µL are unlikely. (Samples in the 300 ng/µL range are often very viscous at room temperature and require heating or vortexing to achieve concentrations above this.)
To ensure smooth processing, we only accept one shipment per quote or order. If an order arrives with fewer samples than the total listed, we will adjust the total order to the appropriate lower amount. We ask that the customer create individual quotes and orders for each shipment.
Because the 48-sample discount only applies to groups of 48 or more samples sent together, please contact us if you would like a budgeting quote for a large number of samples that will be sent in batches of fewer than 48.
Unfortunately, we are unable to purify and culture microbes at our facility at this time. The majority of our services require far more input DNA than a single colony will provide. Agar plate submissions require at least 10⁹ cells per sample, per sequencing pipeline.
We strongly recommend that customers culture samples and verify purity before submissions, with one sample per plate. When submitting agar plates, four-quadrant streak plates are recommended, as they will provide both a quadrant with single colonies for visual verification by the customer and a quadrant with confluent growth for harvesting by SeqCenter.
While we recommend 10⁹ cells at minimum for extraction, we will attempt to extract from all submissions that have some appreciable growth (i.e. more than a few small colonies). If the extraction does not yield a sufficient amount of DNA for sequencing, you will be charged for the cost of the extraction but not for the sequencing services.
Any column-based extraction kit will generally provide favorable results. Bead-based kits are broadly applicable for all services, though more gentle methods such as enzymatic lysis can preserve fragment length, which may be preferable for samples destined for long read DNA sequencing.
Other methods of extraction will also work if care is taken to remove contaminants, such as ethanol and phenol. Some pipelines will be sensitive to other contaminants. Please refer to each pipeline’s specific service page for more details.
While we will attempt to sequence all samples that are sent to us, we highly recommend submitting samples at or above the minimum concentration requirements to increase the likelihood of library preparation success and receive optimal sequencing results. All samples submitted below our concentration requirements will be subject to a $20 fee per failed sample in the event that library preparation is unsuccessful.
For extremely low concentrations with larger volumes, custom handling is available for the Illumina WGS pipeline for an additional fee. Successful custom preparation is not guaranteed, and failed custom preparations will be charged both the custom handling and failed sample fees. Please contact us to discuss if custom handling is feasible for a sample and to make arrangements in advance of shipping the samples.
While we do not have a preference for a specific RNA extraction method, we do recommend a gentle method to preserve transcript length and integrity. We also strongly encourage customers to perform RNA isolation on ice and assess transcript integrity (RIN score), fragment length, and RNA concentration. Please see our Illumina RNA services page for additional information on improving sample and data quality.
All RNA samples are DNase treated and quantified by RNA Qubit prior to library preparation to assess whether there is adequate RNA to proceed. If samples fall below our concentration requirements (at least 1 µg total) post-DNase treatment, this sample will not be processed.
In some instances, we are able to attempt to process samples below our minimum requirements. These require that arrangements be made in advance of submission, as freeze-thaw cycles easily degrade the delicate RNA. If a customer chooses to proceed with library preparation and sequencing of an RNA sample that is below our minimum concentration requirements, the customer will be charged the full cost of services regardless of the resulting data.
All of our standard sequencing services that include library preparation will be given one barcode per sample. (We multiplex many orders together when sequencing, to provide a low per-sample price.) This means that any genomes submitted together as one sequencing sample will be delivered as combined data. If the library preparation includes fragmentation, the genomes may not be able to be separated bioinformatically. In order to be able to re-separate the sequencing data, material meant to be analyzed separately will need to be submitted separately with individual packages ordered for each.
For customer multiplexed and pooled libraries, please see our Illumina Full Flowcell service page or contact us about dedicated Oxford Nanopore and PacBio flowcells. Fully prepared libraries cannot be submitted as individual samples through other pipelines.
For individual samples, we recommend sending microcentrifuge tubes with lids that are sealed shut with parafilm. Please ensure your tubes are correctly and clearly labeled in permanent marker with the sample names associated with your order. Do not write on any tape, parafilm, or stickers used to keep your samples closed as it often gets wiped away or melts in transit during the warmer months.
If sending samples in a 96-well plate, please send only PCR or round bottom 96-well plates that are well sealed. Ship on dry ice to reduce the risk of well-to-well contamination via the seal.
For samples to be extracted, we ask for aspirated cell pellets in sealed microcentrifuge tubes or pre-grown agar plates sealed with parafilm. Agar plates are very delicate and should be well padded and protected for shipment to avoid cracking. When submitting uncultured samples headed for 16S/ITS2 sequencing, please do not submit more than 10mL total material.
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.
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.
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.
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.
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.
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.