Shotgun Metagenomics

16S/ITS Sequencing Shotgun Metagenomics

As the name indicates, shotgun metagenomics is an attempt to capture fragments of eDNA dispersed at random within a microbial community, similar to the spray of a shotgun shell. The acquired DNA is expected to represent the microbial community surveyed and provides an opportunity to assemble whole microbial genomes, capture viral or pathogen interactions that may be missed by 16S/ITS studies, and collect information regarding the abundance of genes within a microbial community to determine putative metabolic activities. While the interpretation of 16S/ITS data is accessible to most, metagenomics poses a more complex challenge. Fortunately, many tutorials exist to help answer most metagenomics questions and represent the diversity of questions that can be addressed with a complex dataset.

At SeqCenter, we utilize our Illumina DNA Library Prep pipeline to perform Illumina shotgun WGS of your entire metagenomic sample. In our data analysis, we provide a basic community composition analysis and an annotated assembly of microbial genomes to help make the data more accessible to the greater scientific community. Shotgun metagenomics can be more costly than the 16S approach but can potentially offer more specific identification, functional gene information, and analytical possibilities for the user. For an example version of our shotgun metagenomics analysis methods, click here, and for an example of the analysis output, click here.

Sample Requirements:

  • Total volume of 30 µL or more
  • Sample eluted into water or dilute Tris/TE buffer
  • Sample concentration greater than:
    • 10 ng/µL measured by Qubit
    • 20 ng/µL measured by Nanodrop

What you will receive:

  • Two fastq files (2 x 151bp) for each sample
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks
  • Shotgun metagenomics analysis includes:
    • Metagenomic Assembly
    • Assembly Annotation
    • Taxonomic Assignment

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
Small Metagenome
Minimum of 6.5M Illumina Reads (1Gbp) with Analysis		 $160.00
Medium Metagenome
Minimum of 32M Illumina Reads (5Gbp) with Analysis		 $300.00
Large Metagenome
Minimum 65M Illumina Reads (10Gbp) with Analysis		 $400.00
Extra Large Metagenome
Minimum of 165M Illumina Reads (25Gbp) with Analysis		 $525.00
*Orders >48 Samples receive 5% discount

We offer convenient DNA extraction services using ZymoBIOMICS DNA extraction kits. This protocol can extract DNA even from 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. We cannot accept soil samples from outside of the US.

Samples requiring DNA extraction can be submitted as aspirated cell pellets shipped frozen on dry ice. A minimum of 109 cells is recommended for all microbial DNA isolation submissions. We cannot culture samples; we encourage you to ensure 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.

16S/ITS Sequencing

The 16S/ITS amplicon approach uses targeted PCR amplification and sequencing of the hypervariable regions of rRNA genes. The results are then binned and queried against a database to provide a taxonomic abundance profile.

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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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