Metatranscriptomics

16S/ITS Sequencing Full-Length 16S (V1-V9) Shotgun Metagenomics Metatranscriptomics

Metatranscriptomics captures RNA expressed within a microbial community, offering a dynamic snapshot of its active biological processes. This RNA reflects both the composition of the community and the metabolic functions being carried out at the time of sampling. Transcripts can be categorized into metabolic pathways (using HUMAnN 3), normalized, and compared across samples—whether within a cohort or between distinct environments. Such surveys provide valuable insights for environmental scientists and pre-clinical researchers by identifying key taxa driving system-level changes, such as those involved in contaminant remediation or disease-associated activity. All metatranscriptomic sequencing services include a functional profiling analysis with normalization.

At SeqCenter, we utilize the Illumina Stranded Total RNA Prep with Ribo-Zero Plus Microbiome kit to generate high-quality RNA libraries. The rRNA depletion included in this prep targets major model organisms, as well as a broader range of bacteria with clinical significance. Beyond that, SeqCenter is also able to design custom depletion probes in partnership with Illumina to ensure optimal depletion of off-target RNA, as needed. If your metatranscriptome is sampled from a host, please consider ordering additional probes to remove host-contaminating RNA (H/M/R hosts are already targeted in the described depletion kit and do not require additional probes). Please contact info@seqcenter.com for additional information on custom probe pools.

Important analysis caveats: Species abundance estimates from metatranscriptomic profiles reflect the proportion of species-specific transcripts, not total transcriptional output. Housekeeping genes with high conservation across taxa may be excluded from this assessment. Species and pathway abundance metrics are influenced by gene copy number, which varies across taxa and may confound apparent pathway activity (Klingenberg & Meinicke, 2017). Additionally, SeqCenter cannot perform differential expression analyses for these packages.

Sample Requirements:

  • Samples shipped frozen on dry ice
  • Total volume of 20 µL or more
  • Sample concentration greater than 50 ng/uL
  • DNAse treated (Recommended, not required)
  • RIN > 6 (Recommended measurement, not required)
  • Host information for bioinformatic depletion requires one of the following:
    • NCBI accession number of host assembly
    • Fasta or GenBank formatted assembly file
    • Denotation of human, mouse, or environmental

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
  • Metatranscriptomic analysis includes:
    • Host and rRNA bioinformatically depleted fastq files
    • Normalized pathway enrichment analyses
    • Microbial taxonomic assignment

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
Small Metatranscriptome
Minimum of 25M Illumina Reads (3.75Gbp) with Analysis		 $290.00
Medium Metatranscriptome
Minimum of 50M Illumina Reads (7.5Gbp) with Analysis		 $340.00
Large Metatranscriptome
Minimum 100M Illumina Reads (15Gbp) with Analysis		 $425.00
*Orders >48 Samples receive 5% discount

Due to the sensitive nature of RNA and RNA-seq projects, we recommend an approach tailored to a project’s organism and experimental setup that is carried out in a uniform manner soon after harvesting.

For customer convenience, we do offer RNA extraction services using the Zymo Quick-RNA Miniprep Kit, which is broadly applicable but may not be optimal for all sample types or projects. This kit enables us to quickly isolate DNA-free RNA from cell pellets as well as homogenized tissue samples. We recommend sending cell pellets that contain at least 107-108 cells, in which the pellet is sizable and clearly visible in the tube. For tissue samples, we ask that you homogenize them as much as possible to enable successful extractions.

This extraction kit is compatible with ZymoResearch’s DNA/RNA Shield, and it should be noted on orders when samples have been treated with this product. More info on the Quick-RNA Miniprep Kit can be found here.

Additional Considerations

Because depletion efficiency is greatly affected by RNA integrity, please see our overall Illumina RNA Services Page for additional tips when working with RNA or preparing for an RNA submission. For more information on our single organism RNA sequencing services, please visit our rRNA Depletion sequencing service page for prokaryotic samples and our mRNA Enrichment sequencing service page for eukaryotic (polyA-tailed) samples.

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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Full-Length 16S (V1-V9)

Supported by PacBio’s CCS technology, SeqCenter’s Full-Length 16S services provides high accuracy reads spanning the V1-V9 of the 16S rRNA gene, maximizing identification and differentiation power for diverse bacterial communities. Results include both sequencing and taxonomic profiling.

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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

Services:

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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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