Simple Fools Guide

guide map


Unless you have direct access to a next-generation sequencing machine, you will need to send your generated cDNA libraries off for sequencing. There are a number of sequencing centers that would be happy to sequence your samples. When selecting a sequencing facility, there are, however, some important things to consider.

1) Whether or not the center performs a quantification step using qPCR and/or an Agilent Bioanalyser (Illumina recommends qPCR). Some centers require this to be done prior to sample submission.

2) Whether or not the center can pool barcoded samples for you or if you have to do that prior to sample submission.

3) How long sequencing takes (ranges from 10 days to several months, depending on their workload).

4) How responsive their customer support is via e-mail or phone.

5) Cost. Some sequencing facilities will also do the sample preparation for you. It is up to you to weigh the options of cost vs. work.

To ensure high quality sequence data, it is usually safer to pick a sequencing center that uses both qPCR and the Agilent Bionalayzer for quality control of cDNA libraries. It may be important to the user that the sequencing takes place in a timely fashion, and it is extremely helpful if the facility's customer support is responsive.


illumina sequencing technology is described here (

Other high-throughput sequencing technologies and systems include:
Applied Biosystems SOLiD (
Roche 454 Life Science (
Helicos Biosciences tSMS (

Tissue   Sequencing   Computer   QC   Assembly   Annotation   Mapping   Expression   SNP

De Wit P, Pespeni MH, Ladner JTBarshis DJ, Seneca F, Jaris H, Overgaard Therkildsen N, Morikawa M and Palumbi SR (2012) The simple fool's guide to population genomics via RNA-Seq: an introduction to high-throughput sequencing data analysis.  Molecular Ecology Resources 12, 1058-1067.