rRNA Depletion

mRNA Enrichment rRNA Depletion

Total RNA sequencing, or rRNA depletion sequencing, enables the analysis of both coding and noncoding transcripts leading to high confidence observations of features such as allele-specific expression, alternative splicing, and novel transcripts. This approach relies on rRNA depletion to remove off-target transcripts. Illumina’s rRNA depletion kits enzymatically degrade the ribosomal sequences before cDNA synthesis, with oligonucleotide probes used to flag the rRNA content. This negative selection leaves behind all other transcripts for sequencing and is designed for a higher tolerance of lower-quality RNA. It results in increased ribosomal content and a larger proportion of intronic sequencing from nascent transcripts. As this protocol requires homologous probes and high RNA integrity for adequate depletion, it can be less efficient than polyA tail capture depending on probe specificity; however, it is irreplaceable for organisms lacking polyA tailed transcripts and for reliably examining noncoding transcripts.

For rRNA depletion sequencing, SeqCenter uses Illumina’s Stranded Total RNA Prep with Ribo-Zero Plus Microbiome kit to generate unbiased high-quality RNA libraries. The two depletion kits included in this prep are targeted towards major model organisms as well as a broader range of bacteria with clinical significance. Beyond that, SeqCenter is able to design custom depletion probes in partnership with Illumina to ensure optimal depletion in your sample if your project’s organism is not covered within the standard range. For more information, see the depletion section below.

We strongly recommend visiting our overall Illumina RNA services page for additional considerations when planning any RNA experiment and encourage all customers to check out the tips for handling RNA and guidance when assessing sample quality for submission.

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)

What You Will Receive:

  • Two fastq files (2 x 150bp) for each sample
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
12M Paired End Reads (1.8 Gbp)
Example uses: Bacteria		 $250.00
25M Paired End Reads (3.75 Gbp)
Example uses: Yeast		 $275.00
50M Paired End Reads (7.5 Gbp)
Example uses: Larger fungi, fruit flies, algae		 $320.00
100M Paired End Reads (15 Gbp)
Example uses: Mouse, human, simple metatranscriptomes		 $400.00
200M Paired End Reads (30 Gbp)
Example uses: Transcriptome assembly, diverse metatranscriptomes		 $550.00
*Orders >48 Samples receive 5% discount

We offer two levels of RNA analysis packages, Basic and Intermediate, which can help our customers to start analyzing their differential expression studies. These packages are a great starting place for burgeoning bioinformaticians, but they will not pick up non-coding RNA and are not intended for metatranscriptomes. For more information, please visit our RNA Analysis services page.

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.

Depletion, Standard Probe Target Information, and Custom Probes

SeqCenter’s RNA sequencing services with rRNA depletion rely on both Illumina’s proven Ribo-Zero Plus kit and the expanded range Ribo-Zero Plus Microbiome kit to provide depletion for a broad range of common microbial targets and their hosts. Below is more information on the specific targets that these kits are designed against and custom probe options if yours is not closely related any on the lists.

Illumina’s main rRNA depletion is proven efficient against B. cereus, B. subtilis, E. cloacae, E. coli, S. epidermis, as well as the organisms in ATCC MSA-2002 and MSA-2006. Below is a list of the specific design targets for the probes in this kit.

  • Human cytoplasmic rRNAs: 28S, 18S, 5.8S, 5S
  • Human mitochondrial rRNAs: 12S, 16S
  • Human beta globin transcripts: HBA1, HBA2, HBB, HBG1, HBG2
  • Mouse & Rat rRNA: 16S, 28S
  • Gram(-) Bacterial rRNAs: Escherichia coli: 5S, 16S, 23S
  • Gram(+) Bacterial rRNAs: Bacillus subtilis: 5S, 16S, 23S

For more information on the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus, please see Illumina’s data sheet for the prep and depletion kits.

The Microbiome kit expands the capture probe set and was designed against 5S, 16S, and 23S rRNA from diverse microbial species, including common adult and infant gut bacteria, ATCC MSA-2002, MSA-2005, and MSA-2006. Below is a compiled list of the specific targets included in the three ATCC mixes:

  • Acinetobacter baumannii (ATCC 17978)
  • Acinetobacter johnsonii (ATCC 17909)
  • Bacillus pacificus (ATCC 10987)
  • Bacteriodes fragilis (ATCC 25285)
  • Bacteroides vulgatus (ATCC 8482)
  • Bifidobacterium adolescentis (ATCC 15703)
  • Clostridioides difficile (ATCC 9689)
  • Clostridium beijerinckii (ATCC 35702)
  • Corynebacterium striatum (ATCC 6940)
  • Cutibacterium acnes (ATCC 11828)
  • Deinococcus radiodurans (ATCC BAA-816)
  • Enterobacter cloacae (ATCC 13047)
  • Enterococcus faecalis (ATCC 47077)
  • Enterococcus faecalis (ATCC 700802)
  • Escherichia coli (ATCC 700926)
  • Fusobacterium nucleatum (ATCC 25586)
  • Helicobacter pylori (ATCC 700392)
  • Lactobacillus gasseri (ATCC 33323)
  • Lactobacillus plantarum (ATCC BAA-793)
  • Micrococcus luteus (ATCC 4698)
  • Neisseria meningitidis (ATCC BAA-335)
  • Porphyromonas gingivalis (ATCC 33277)
  • Pseudomonas paraeruginosa (ATCC 9027)
  • Rhodobacter sphaeroides (ATCC 17029)
  • Salmonella enterica subsp. enterica (ATCC 9150)
  • Schaalia odontolytica (ATCC 17982)
  • Staphylococcus aureus (ATCC BAA-1556)
  • Staphylococcus epidermidis (ATCC 12228)
  • Streptococcus agalactiae (ATCC BAA-611)
  • Streptococcus mitis (ATCC 49456)
  • Streptococcus mutans (ATCC 700610)
  • Yersinia enterocolitica (ATCC 27729)

For more information on the Microbiome Depletion Kit, please see Illumina’s data sheet for this kit.

Custom depletion probes can be used in place of the Microbiome add-in, and alongside the standard Ribo-Zero Plus set, to ensure optimal depletion for specific organisms. SeqCenter works with Illumina to design custom probes using either an annotated reference genome or specific ribosomal target sequences. Probes are then ordered from IDT and typically arrive within one to two weeks for use.

For additional information on custom probe pools, including a quotation, please contact us.

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.

mRNA Enrichment

PolyA enrichment positively selects for transcripts with polyadenylated tails and is often referred to as mRNA sequencing. This method provides a clean way of sequencing only on-target fragments but is only applicable for organisms that create polyA-tailed transcripts. Because of this, polyA enrichment sequencing is generally the choice for eukaryote-only studies.

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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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