Long RNA Sequencing Services

Ocean Ridge Biosciences (ORB) provides comprehensive services to analyze mRNA and long non-coding RNA using sequencing with Illumina platforms. Depending on a project's goals and requirements, ORB’s services may include pre-project planning and consultation, RNA extraction, library preparation, sequencing, processing of raw sequence reads, in-depth analysis of processed data, and post-project support of the publication of results as well as patent and FDA application submissions.

The established applications that ORB is equipped to handle include classical mRNA profiling, long non-coding RNA analysis, and alternative splicing analysis. Novel applications that have been enabled by long-read Illumina sequencing include allele-specific RNA expression, RNA editing, RNA polymorphism analysis, and the detection of gene fusion events; ORB’s customized bioinformatic services enable these next-generation apps.

Long RNA library preparation technology has advanced dramatically since the introduction of the HiSeq 2000 in 2010, and ORB has kept up with the advances in order to deliver state of the art sample preparation services to our clients. ORB’s long RNA library preparation capabilities include removal of rRNA and globin cDNA sequences from sequencing libraries, working with as little as 30 picograms of intact RNA and 500 picograms of partially degraded RNA, and the preparation of sequencing libraries from prokaryotic species.

ORB's long RNA sequencing workflow
Figure 1. ORB's long RNA sequencing workflow.

 

To obtain more information on specific topics regarding long RNA sequencing — please click on the links below:

Sequencing
Platform
Project
Capabilities
Processing
Options
Bioinformatics

 

ORB's Project Capabilities

With more than 5 years experience in sequencing-based long RNA profiling services, ORB has expertise in a wide variety of long RNA sequencing experiments. ORB can perform RNA extraction starting from human, mouse, and rat tissue, as well as cultured and flow-sorted cells (see ORB’s biological sample processing page for more information). ORB can also prepare sequencing libraries from purified RNA samples provided by clients, even those that are partially degraded or very low input. ORB routinely processes RNA samples with mass as low as 30 picograms. ORB scientists enthusiastically participate in challenging collaborative projects that involve testing novel processes or working with difficult samples such as immunoprecipitated RNA, fixed cells, or non-cellular RNA. ORB can also directly sequence prepared libraries. Common project types where ORB provides long RNA profiling experience and expertise include:

  • Disease research using human postmortem and surgically ressected tissues
  • Identification of drug targets and key pathways using rodent disease models
  • Biomarker discovery using blood samples collected in PaxGene tubes or PBMCs
  • Analysis of subcellular RNA fractions in model organisms
  • Characterization of cell-type specific expression and drug response in flow-sorted and LCM cells
  • Sequencing of immunoprecipitated RNA from bacTRAP experiments
  • Expression profiling in domesticated farm animals and agriculturally important plant species
  • Analysis of both graft and host expression in xenograft models
  • Characterization of microflora and host gene expression in stool samples
  • Identification of long non-coding RNAs associated with neurological disease

Illumina Sequencing Platform
ORB performs sequencing using the NextSeq 500, HiSeq 2500, and HiSeq 4000 depending on the project size and specific application. Please visit our Illumina sequencing page for details about these sequencing instruments, metrics, and the typical applications at ORB. 

Processing Options - Library Preparation
ORB utilizes the latest library preparation kits from Nugen, llumina, and Clontech to cover the range of projects from ultra-low (30 pg) to standard input (2 ug) and from severely degraded to intact RNA. Polyadenylated mRNA can be specifically targeted for sequencing or conversely, non-target RNA species such as ribosomal and globin RNA can be specifically depleted. Both strand-specific and unstranded libraries can be produced depending on the application. Please see Table I, below for a list of some of the kits validated at ORB. A sample process flow for standard and low input library preparation using kits provided by Illumina and Nugen is depicted in Figure 2.

 

ORB will gladly support your RNA-Seq projects with challenging or unusual requirements. Whether it is sequencing of immunoprecipitated RNA, sequencing of RNA from subcellular compartments, or ultra-low input situations we welcome the opportunity to work with you! We look forward to hearing from you about your unique project.


Table I. Validated Library Prep Kits in Use at ORB
Input Mass
(ng)
RNA Quality Appropriate Kit
100-2000 Intact to Minor Degradation Illumina TruSeq RNA Sample Prep Kit v2
150-2000 Intact to Minor Degradation Illumina TruSeq Stranded mRNA
100-2000 Moderate Degradation Illumina TruSeq Stranded Total RNA w/ RiboZero
100-2000 Moderate Degradation Illumina TruSeq Stranded Total RNA Blood
100-2000 Moderate Degradation ScriptSeq Complete Gold (Epidemiology or Bacteria)
0.5-50 Moderate Degradation NuGen Ovation RNA-Seq Sys. 2 w/ Illumina TruSeq DNA Nano
0.03-10 Intact to Minor Degradation Clontech SMART-Seq v4 Ultra Low Input w/ Illumina Nextera
0.25-10 Moderate Degradation Clontech SMART-Seq

Stranded Total RNA - Pico Input Mammalian Kit


Processing Options - Sequencing
The length of reads, configuration (single or paired), and the number of reads is determined by the sequencing application, while the choice of sequencer is primarily determined by the desired length of the read and the batch size (Table II). For example, 16 million single-end reads of 75-100 nucleotides is sufficient for reliable mRNA counting by sequencing1 . This can be achieved by loading 12 samples per lane on the HiSeq 2500 or 20 samples per run on the NextSeq 500. Improved mapping and detection of low abundance mRNAs can be achieved by approximately doubling the quantity of read per direction and using a paired-end approach (Table II).

Figure 2. Examples of low and standard input library preparation procedures utilizing the Illumina TruSeq Nano HT DNA Library Preparation Kit in the final steps. One of many RNA-Seq library preparation methods available at ORB.

For studies of mammalian alternative splicing, long non-coding RNA, and RNA polymorphism detection with an even greater depth and longer reads are desirable. Both 100 and 150 nucleotide PE reads are suitable for alternative splicing analysis, and the HiSeq 4000 provides the most cost-effective price per nucleotide sequenced in its 150PE configuration. At the same time the HiSeq 4000 can handle up to 80 samples per run versus 7 samples on the NextSeq and 64 samples on the HiSeq 2500 (Table II). Please see Figure 3 to view an outline of the process flow for library quality assessment through sequencing on the Illumina HiSeq 2500 at ORB.

ORB will accept your direction for choice of sequencer, as well as read configuration and quantity, or ORB staff will gladly plan a customized process to minimize the cost and time required to meet your study objectives; contact us to receive your custom quote.

 

Table II. Recommended Sequencing Configuration– Illumina Platform

Application Reads (per direction)

Read Configa,b

Sequencer Samples per laneb Samples per full runb
Mammalian Gene-level mRNA profiling 16M 100 SE
75 SE
HiSeq 2500
NextSeq 500
12
NA
192
20
Mammalian Gene-level mRNA profiling with improved mapping and detection of low abundance mRNA 30M 50 PE
75 PE
HiSeq 2500
NextSeq 500
6
NA
96
10
Mammalian Alternative Splicing, RNA polymorphisms, long ncRNA3 45M 100 PE
150 PE
150 PE
HiSeq 2500
NextSeq 500
HiSeq 4000
4
NA
5
64
7
80
Vertebrate De Novo
Transcriptome Assembly
90M 150 PE
150 PE
NextSeq 500
HiSeq 4000
NA
2
3
32
Bacterial Gene Expression Profiling 10M 75 SE NextSeq 500 NA 32
  1. Numeric value in read configuration is nucleotides; SE - single-end, PE - paired-end.
  2. For background information please download Illumina specifications for the HiSeq 2500, NextSeq 500, and HiSeq 4000.

 

Figure 3. Process-flow for library quality assessment and sequencing on the Illumina HiSeq 2500.


References

  1. Chhangawala, S., Rudy, G., Mason, C. E., & Rosenfeld, J. A. (2015). The impact of read length on quantification of differentially expressed genes and splice junction detection. Genome biology16(1), 131.