16S/ITS Sequencing

16S/ITS Sequencing Shotgun Metagenomics

The 16S and ITS amplicon approaches use PCR to amplify the hypervariable regions of rRNA gene found in nearly all bacteria and archaea or fungi, respectively. Conserved regions of rRNA allow for consistent and accurate alignment of sequenced data, while the variable “fingerprint” regions allow for the classification of amplicon sequence variants (ASVs). Identified ASVs are taxonomically assessed using the full-length Silva-138 (16S) and Unite-8 (ITS) databases. The result of this workflow is the taxonomic profiling of a microbial community to determine the abundance of unique taxa in each system. As this technology has grown, representative databases like the Silva database have served as essential resources for properly classifying microbes.

At SeqCenter, we utilize Zymo Quick-16S Plus or Quick-ITS Plus Library Prep methods to selectively amplify either the V3/V4 region (bacteria/archaea) or ITS2 region (fungi) of your metagenomic sample, with reads spanning roughly 600 bp and 480 bp, respectively. All 16S and ITS services come with taxonomic analyses and assessments of alpha and beta diversity. While the applications for this technology are great, the reliance on precompiled databases poses challenges for poorly classified communities not represented in the available databases. This has led to a surge in shotgun metagenomics studies which aim to capture all available genomic information for a given system, including gene content and increased specificity of identification, though it is more costly to attain comparative amounts of depth.

For an example version of our 16S methods including primers used, click here; for the ITS methods, click here; and for a downloadable example of the 16S analysis, click here.

Sample Requirements:

  • Total volume of 20 µL or more
  • Sample eluted into water or dilute Tris/TE buffer

What You Will Receive:

  • Two fastq files (2 x 301bp) for each sample
  • Sequencing stats summary
  • Materials and methods summary
  • ASV representative sequence and raw counts file
  • Data shared via Globus within 2 weeks
  • Analysis reports include:
    • Species abundance tables
    • Alpha and beta diversity
    • Taxonomic assignment
    • Diversity barplots by taxonomic rank

Packages and Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
20k Reads (10k Read Pairs)
Either V3/V4 or ITS2 Region		 $45.00
50k Reads (25k Read Pairs)
Either V3/V4 or ITS2 Region		 $55.00
100k Reads (50k Read Pairs)
Either V3/V4 or ITS2 Region		 $75.00
*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.

16S and ITS2 samples requiring DNA extraction can be submitted as frozen environmental samples or as aspirated cell pellets shipped frozen on dry ice.

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.

Shotgun Metagenomics

Our Shotgun Metagenomics packages sequence randomly spread eDNA fragments within a sample and attempt to create metagenomic assemblies for the members of the community. This can provide a survey of functional genes within the community and a plethora of analytical opportunities.

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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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