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cDNA Sequencing

Transcriptome sequencing is a term that encompasses experiments including mRNA transcript–expression analysis (full–length mRNA, expressed sequence tags (ESTs) and ditags), novel gene discovery, gene space identification in novel genomes, assembly of full–length genes, single nucleotide polymorphism (SNP), insertion–deletion and splice–variant discovery, as well as analyses of allele–specific expression and chromosomal rearrangement. The combination of long, accurate reads and high throughput makes 454 Sequencing analysis on the Genome Sequencer FLX ideally suited to detailed transcriptome investigation.

Guidelines to cDNA Sequencing

The number of transcriptome sequencing runs required depends on several factors, including the following:

  • The purpose of the experiment
  • The completeness of mRNA species in the sequenced sample
  • The estimated gene count
  • The sequence yield (total number of reads) per run
  • The average length of raw reads
  • The normalization of cDNA library
  • The quality of the mRNA sample

We recommend the following equation to estimate the required number of runs:
Total number of bases required (Mb) = (Estimated gene count in the sample to be sequenced) x 40,000
Or, for every 8,000 genes in a sequenced RNA sample, we recommend one full GS FLX Titanium sequencing Run (approximately 1 million reads with an average length of 350 bases). To obtain wider dynamic range in read count, add appropriate number of runs as desired.

Usage of pooled and normalized cDNA libraries is recommended for gene discovery and genome annotation. Normalized samples are likely to produce higher sensitivity and better gene coverage than non-normalized cDNA libraries.
Non-normalized cDNA libraries are used for measuring relative gene expression using read counts.

RNA Sample Requirements

The sample RNA should be:

  • total amount of RNA > 200 ng
  • quantitated by Ribogreen
  • sample volume > 19 μl
  • pure (OD 260/280 > 1.8)
  • DNA free
  • enriched in RNA of interest. For example, if the RNA of interest is mRNA, remove ribosomal RNA prior to proceeding with the procedure
  • quality assessed on an RNA 6000 Pico Chip on the Agilent 2100 Bioanalyzer instrument. A typical RNA sample will produce a smear that ranges from 0.2 kb to 7 kb.

This protocol is not designed for preparing small RNA molecules, for example snoRNA, microRNA, tRNA, etc,.