Frequently asked questions

Please see our Sample Preparation Guide (below) for detailed information about how samples should be prepared.

  • Please see our Shipping page for detailed information about how to ship your samples to us.
  • DNA extraction: many column-based extraction kits provide good results, as do other methods if care is taken to remove contaminants. Our library prep methods work best with buffers containing less than 1mM EDTA.
  • RNA extraction: we recommend Qiagen RNeasy kits with DNase treatment, or similar.
  • Metagenomic DNA samples: we recommend a physical lysis method such as bead beating to preserve community diversity.
  • Long read sequencing: we strongly recommend sending raw samples for us to extract nucleic acids composed of longer fragments for better service.

We recommend Qubit or other fluorescent dye-based measures of nucleic acid quantification, or Bioanalyzer/Tapestation. Nanodrop is also acceptable, though we ask that you aim to supply well above the requested 10ng/ul minimum as this technology tends to overestimate concentrations by as much as 10-fold. When Nanodrop and Qubit values are discordant this is often an indication of contaminants such as phenol or ethanol, which can sometimes interfere with NGS workflows.

It depends on your research question! Here are a few guidelines:

  • Variant calling, or mutation detection in comparison with a reference genome, typically requires a minimum of 30x mean coverage. Converting this to our offerings, multiply [desired coverage] x [genome size, in Mb]. 
  • For de novo hybrid assembly, we recommend at least 50X short reads and 30X long reads.

For genome resequencing projects, we strongly encourage including your wild-type strain along with your test samples.

Sequencing data for most sample types is available within 14 business days of receipt of your samples, if not sooner. Bioinformatics deliverables are typically available within 7 business days of receiving your sequencing data. You will receive an email confirmation of receipt and a projected delivery date for your order.

We will upload your data to a personal folder on Box.com or AWS S3, (your choice) and provide you with a link to the files for your order. The data will be available to you for one year.

Within your folder, you will find compressed FASTQ files for each sample (2 per sample for paired end reads), a README of methods, sample QC files, and any analysis services you requested in a separate directory.

Credit Cards

We welcome payments via all major credit cards. Please note, credit card payments are subject to a processing fee that helps us cover part of the costs charged by our card processor, Stripe.

Purchase Orders

For organizations wishing to use purchase orders, we invite you to register SeqCoast within your organization’s procurement system. For assistance with registration, please contact our finance team at finance@seqcoast.com

Wire Transfers

Wire transfers are accepted and include a surcharge of $30.00 to cover incoming bank fees. For international transfers where the institutional policy is to deduct outgoing wire fees from the payment amount, an additional $30.00 surcharge will apply.

For any further assistance or clarification on making payments, please reach out to finance@seqcoast.com.

TL;DR

16S is used to profile microbial communities. Full-length 16S sequencing (Nanopore) yields higher resolution of species and strains compared to V3/V4 sequencing (Illumina).

Long version:

16S rRNA sequencing is used to examine bacterial species diversity and abundance of a microbial community. The 16S rRNA gene is a well-established marker for bacterial species, and it consists of both conserved regions and hypervariable regions (V1-V9). Primers bind to the conserved regions, and the hypervariable regions are amplified, sequenced, and analyzed.

Because the entire 16S sequence is about 1500bp long, only a subset of the sequence can be revealed using Illumina sequencing. We use primers 341F and 806R to amplify the V3/V4 hypervariable regions, and we perform 2x300bp paired end sequencing. This sequence length allows the paired reads to overlap, maximizing accuracy. Still, Illumina sequencing often fails to resolve species or strain-level differences. If this level of detail is important for your project, use our Nanopore full-length 16S service to sequence and analyze the entire 16S gene!

Our standard protocols are designed to accommodate a broad range of biological samples, including bacteria, fungi, algae, and various cell lines, given that the recommended inputs are provided. While we strive to ensure the highest quality and compatibility, it’s important to note that we cannot guarantee flawless performance for every species or sample type due to the inherent variability in biological materials.

We offer custom processing for complex samples such as stool, soil, filters, and others that may require specialized workflows. Please contact us to get a quote on your next custom project!

We accept all samples in PCR tubes, 1.5 mL tubes, or 96-well plates.

Samples in Tubes

When sending samples in tubes, we recommend wrapping tube lids in Parafilm when possible, and packing samples with plenty of foam or bubble wrap for protection. This helps prevent tubes from popping open or becoming crushed during transit.

Samples in 96-Well Plates

For large orders, it is often easier to send 96-well plates. We recommend using PCR plates that are well-sealed with either foil seals or plastic heat-seals. We strongly suggest shipping the plates on dry ice if possible. We find that sometimes the shaking and sloshing from the shipping process defeats even the strongest plate seals, and keeping the samples frozen helps prevent well-to-well contamination.

Additionally, in your sample manifest, it is important to indicate which sample is in which well. The best way to do this is to add a column labeled “Well ID” to your sample manifest before uploading.

Sample Preparation Guidelines

Nucleic Acid Samples

Service Concentration* Volume Buffer Shipping Considerations
Short read sequencing, Illumina
>10 ng/µL
>30 µL
nuclease-free water or dilute TE (1:10)
Can be shipped at room temperature.
Long read sequencing, Oxford Nanopore
>50 ng/µL
>60 µL
nuclease-free water
Should be shipped with ice packs or on dry ice.
Poly-A Enrichment RNAseq, Illumina
>25 ng/µL RNA
>20 µL. Please include a second aliquot (~5 µL) for sample QC.
nuclease-free water or dilute TE (1:10)
RNA should be shipped overnight on dry ice.
Ribodepletion RNAseq, Illumina
>25 ng/µL RNA
>20 µL. Please include a second aliquot (~5 µL) for sample QC.
nuclease-free water or dilute TE (1:10)
RNA should be shipped overnight on dry ice.
16S Amplicon (Illumina or Nanopore)
>10 ng/µL
>30 µL
nuclease-free water
Can be shipped at room temperature.
Full Flow Cell Runs, Illumina
Prepared and pooled libraries with a final concentration of at least 2 nM.
RBS + Tween
Can be shipped with ice packs or on dry ice.
Full Flow Cell Runs, Nanopore
Same as long read Nanopore
nuclease-free water
Should be shipped with ice packs or on dry ice.

*We recommend quantifying by Qubit, as Nanodrop can overestimate concentrations >10-fold if contaminants are present.

We highly recommend eluting samples in nuclease-free water or diluted TE, as excessive EDTA can interfere with with library prep. Extraction contaminants (salts, phenol, polysaccharides, etc.) can cause sample failure.

Services that include DNA Extraction

Service Sample Type Shipping Considerations
Hybrid whole genome sequencing, Illumina/Nanopore OR Strain typing, Illumina
Frozen cell pellets (50 - 100 mg), all supernatent removed; OR Sealed, pre-grown agar plates - must have enough growth to allow us to easily collect 50 - 100 mg of colonies.
Ship frozen cell pellets on dry ice. Ship agar plates with ice packs or at room temperature.

Looking for detailed guidance on selecting the right sequencing approach for your science? Our consultation services provide expert advice tailored to your project's needs.