RNA Analysis

Assembly and Annotation RNA Analysis Haploid Variant Calling Custom Analysis

The goal of many RNA sequencing projects is to determine how genes are differentially expressed between experimental groups. With the ever-decreasing cost of sequencing and increasing flowcell capacities, processing large amounts of RNA-seq data can require vast computational resources not readily available to the average researcher and may hinder researchers from starting their analyses.

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

Basic RNA Analysis

The first level of analysis we offer, Basic Analysis, maps RNA sequencing reads to a provided annotated reference genome. Read mapping and counting can be computationally expensive, so this package is ideal for customers who wish to perform their own comparisons and more in-depth analysis, but may lack the computational resources necessary to produce the raw counts in a reasonable amount of time.

Raw counts of CDS annotated genes are returned in table format, with multi-mapping reads discarded. Examples of methods can be found here for prokaryotic samples and here for eukaryotic samples. For experiments with multiple organisms, we standardly recommend performing independent mapping for each organism.

Requirements:

  • Data must be sequenced through an Illumina RNA pipeline.
  • An annotated assembly that is highly identical to the sample is required, in GenBank format (.gb, .gbk, .gbff). Other formats are not accepted.
    • For assemblies uploaded to NCBI, accession numbers are also accepted. Please verify that the correct version is specified (GenBank vs RefSeq) and reference with the correct accession number (GCA vs GCF).
  • Multi-organism analyses may incur additional charges and require additional considerations that should be discussed prior to sample submission.

Intermediate RNA Analysis

The second level we offer is Intermediate RNA Analysis, which expands on the Basic package. After mapping and counting, the pipeline normalizes the raw CDS counts and makes pairwise comparisons between experimental group means. If the reference organism is in the KEGG Pathway Catalog, pathway information is included in the output. Counts are normalized using edgeR’s Trimmed Mean of M’s method (TMM) before statistical analysis is performed. An overall PCA plot for general assessment of the samples is provided, along with high-level heatmaps and full and filtered lists of the differentially expressed genes for each comparison.

This package is most effective with multiple experimental replicates for each group. Multi-dimensional analyses are not supported.

Requirements:

  • Data must be sequenced through an Illumina RNA pipeline.
  • An annotated assembly that is highly identical to the sample is required, in GenBank format (.gb, .gbk, .gbff). Other formats are not accepted.
    • For assemblies uploaded to NCBI, accession numbers are also accepted. Please verify that the correct version is specified (GenBank vs RefSeq) and reference with the correct accession number (GCA vs GCF).
  • Multi-organism analyses may incur additional charges. Please contact us to discuss.
  • Each pairwise comparison between groups to be made must be listed. (Ex. Treatment 1 vs Untreated)
  • For pathway analysis to be included, the requested reference must be included in the KEGG Pathway Catalog.

Controlling for Batch Effect

Sequencing data, and RNA analysis by extension, is very sensitive to batch effects. Because of this, if additional sequencing or replicates will be added to an analysis, we strongly recommend that samples be added in a symmetrical fashion across treatment groups. (Ex. Each round of library preparation and sequencing should contain representatives from each group in equal numbers.) This type of symmetric batch effect can be controlled for during intermediate analysis and will minimize skew resulting from differences in batches.

Assembly and Annotation

As the volume of generated sequence data continues to expand, the scientific community has a responsibility to ensure that the data accurately reflects the genomic information of the host organism. This analysis is valid for our Illumina, Oxford Nanopore, and PacBio sequencing packages for homogeneous samples, with some options for metagenomic samples.

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Haploid Variant Calling

Mutations can be captured through whole genome sequencing and subsequent variant calling to identify SNPs, indels, and other mutations within monoclonal, haploid samples. Analysis is valid for our Illumina DNA sequencing packages and Oxford Nanopore WGS packages.

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Custom Analysis

The needs and demands of the scientific community rapidly adapt to innovations in technology and literature. This analysis is valid for our Illumina, Oxford Nanopore, and PacBio sequencing packages.

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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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