Introduction

RNA-Seq is a widely used technique for studying gene expression levels within cells or tissues. It involves sequencing the entire mRNA population present in a given sample. Gene expression levels can then be compared between groups of samples from different experimental conditions.

 

Workflow

An overview of the RNA-seq pipeline is shown below. Key steps include sequence quality evaluation using FastQC, read trimming and filtering using Trimmomatic, additional read filtering using SortMeRNA, read mapping using HISAT2, and differential expression analysis using DESeq2.

The final results of the pipeline are outlined in blue.

1. An HTML formatted sequencing quality report produced by FastQC.
2. Tables with the results of the DESeq2 analysis in both CSV and XLSX formats. These tables include both the raw read counts used as input data to DESeq2 and the differential expression analysis results.
3. A final analysis report in PDF format containing a description of the methods used and some additional QC and summary figures.

Pricing

Our current price for this analysis is $60 per sample.

As a reminder, it is best practice to plan for a 3% to 5% yearly rate increase when proposing studies and submitting grants.

References

Please refer to these resources and tools that form an integral part of the pipeline process.

1. FastQC – A quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
2. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014 Aug 1;30(15):2114-20. doi: 10.1093/bioinformatics/btu170
3. Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscrip- tomic data. Bioinformatics. 2012 Dec 15;28(24):3211-7. doi: 10.1093/bioinformatics/bts611
4. Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019 Aug;37(8):907-915. doi: 10.1038/s41587-019-0201-4
5. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, Whitwham A, Keane T, McCarthy SA, Davies RM, Li H. Twelve years of SAMtools and BCFtools. Gigascience. 2021 Feb 16;10(2):giab008. doi: 10.1093/gigascience/giab008
6. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014 Apr 1;30(7):923-30. doi: 10.1093/bioinformatics/btt656
7. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi: 10.1186/s13059-014-0550-8

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