For application or experimental design advice please contact Karen Moore (K.A.Moore@exeter.ac.

uk)
For further information, contact Konrad Paszkiewicz (K.H.Paszkiewicz@exeter.ac.uk)

University of Exeter DNA Sequencing Service

Illumina technology: sequencing by synthesis
The basic principle

Step1: Library preparation Step 2: Cluster on flowcell Step 3: Sequencing by synthesis
Fragmentation (RNA cDNA) Attach DNA to flowcell surface Extend first base, read fluorescent
End repair, add A overhangs Form DNA bridges signal and de-block
Ligate adapters Amplify bridges into clusters Repeat cycle above extend strand
Size select fragments Anneal sequencing primer Generate base calls
Step 4: Analysis
Base calling is run automatically based on cluster intensity and cluster position

Sequencing can be single-ended, paired-end or multiplexed if barcodes are present
Instrumentation: Illumina HiSeq 2500
Robust base calling reduces sequence context errors
Good sequencing of repetitive regions and homopolymers
2 modes of operation; high throughput (2 lanes) or rapid run (8 lanes)
2 flowcells can be imaged simultaneously

Single end or paired end reads of 50, 100 or 150 nucleotides
Up to 200 million reads per lane enables multiplexing
5 human genomes at 30x coverage sequenced in 11 days
Karen Moore, Audrey Farbos, Paul ONeill and Konrad Paszkiewicz
Getting the most from RNAseq
http://ess-wiki.exeter.ac.uk/ess/Exeter_Sequencing_Service/
Active mRNA Translation-seq

Sequence ribosome-protected mRNA fragments
(ribosomes active in a cell at a specific time point)
Investigate translational control, measure gene
expression, and predict protein abundance.
Available for mammalian and yeast samples
Molecular indexing of RNA provides an
absolute digital measure of gene expression

96 adapters that include 8-nucleotide barcode tags are present in molar excess
each end of cDNA is ligated to single labelled adapter at random
96 x 96 possible combinations (9,216) given by the first 8 bases in each PE read
sample specific barcodes can be included and read during a multiplexing index read
reads from PCR duplicates are readily distinguished from ESTs
Determine the lower limit of detection and assess the
fold change response in RNAseq using external controls

External RNA Controls Consortium (ERCC) synthetic RNAs (Life Technologies)
92 synthetic poly-adenylated RNAs spanning a 10
6
-fold concentration range
spiked into the input RNA sample to serve as internal standards
two formulations differ in the proportions of transcripts (4 subgroups):


After applying appropriate normalization and filtering to the expression data (RPKM,
CTs, RFUs, etc), plot the signal for each ERCC transcript against its known molar
concentration or amount, and use linear regression to determine the best-fit line. This
plot is often described as a dose-response curve.
The dynamic range can be measured as the difference between the highest and
lowest concentration of ERCC transcript detected in each sample
Compare the observed fold-change ratio (Mix 1:Mix 2) to the
expected ratio provided in ERCC_Controls_Analysis.txt.
Determine the concordance of the ratios by linear regression to
assess the platform performance.