NGS Transcriptomic Workflows

Hugh Shanahan & Jamie al-NasirRoyal Holloway, University of London

Transcriptome – total sequence and abundance of RNA generated by a cell

RNA is transcribed from DNA Genome is fixed for a organism Transcriptome is dynamic

Variation between tissues Variation over time

RNA transcripts are 1,000’s-10,000 bases in length

Setting the scene

Interested in How many copies of a particular transcript

are there What is the sequence

- sequence comes from genome but alternative splicing means a transcript may not just be a contiguous block of DNA

Size of transcriptome will vary between species

Fragment transcripts into shorter pieces (reads) 100-300 bases longs

Have many overlapping reads

Amplify (make lots of copies of) the short reads

Can sequence these short reads and then assemble them to reconstruct transcripts.

Size of data set depends on size of transcriptome but also amount of fragmentation (sequencing depth)

Can either assemble with a reference genome or de novo (very hard)

Sequencing steps

NGS Workflow

NGS Workflow

File formats have been updated to binary – used to use flat text so sizes were huge (Reference Genome – 39 Gbyte -> 0.8 Gybte)

Raw image data is actually discarded Discussions focusses on assembly and down-stream

analysis Much of this data is deposited in the Sequence Read

Archive (SRA) We’ve papered over everything that happens before

sequencing – i.e. the biochemical steps carried out This is highly variable These steps are not properly annotated

Final points