1. Introduction to Illumina Sequencing
Day 1, Video 2
Overview of Next-gen sequencing
Introduction to Illumina sequencing
Multiplexing
Sequencing run statistics

2. Next-Gen Sequencing Millions of reactions performed in parallel
Shorter read lengths, higher error rate
Sample/library prep is required
Many different approaches
Illumina sequencing-by-synthesis (Solexa technology)
Roche 454 pyrosequencing
AB SOLID color-based sequencing by ligation
Ion Torrent semiconductor sequencing
Single-molecule sequencing (PacBio, MinION, etc)

3. Some general terminology
SR: single-read sequencing, sequence from only one end
PE: paired-end sequencing, sequence from both ends
Adapters: DNA added to the ends of DNA/RNA fragments to be sequenced. The adapters allow the DNA/RNA to attach to the flowcell
Index/barcode: used interchangeable to indicate sequence identifier for multiplexing
PhiX: commercially available genomic library of PhiX bacteriophage DNA, commonly spiked into libraries

4. Steps to Illumina sequencing
Library construction
Fragment, attach adapter DNA
Cluster generation
Add to flow cell
Bridge amplification
Sequencing
Single base at a time, imaging
Data analysis
Images transformed into basecalls and ‘reads’

5. Illumina sequencing SBS chemistry video

6. Clustering, the first step to sequencing

7. Sequencing by Synthesis overview

8. The importance of cluster density
Well-spaced clusters easier to call
Densely-packed clusters difficult to call
Illumina reports “optimal” cluster density for each platform
pM amounts of libraries are used for sequencing
Accurate QC and quantification are essential!

9. Anatomy of a library P5 and P7 ends of adapters bind to flow cell
DNA insert typically ranges bp (<1kb)
Different methods of indexing
Inline (part of the insert) – any level of multiplexing
Single index read (≤96)
Dual index reads (384+)

10. Multiplexing – single index read

11. Multiplexing – dual index readshf

12. Some terminology
Clusters (raw): number of clusters detected through imaging
Reads: the number of reads – some people refer to a cluster as a read (a DNA molecule), others refer to the number of sequences so for PE data this is 2 x DNA molecules
% passed-filter (%PF): % of clusters or reads that pass a chastity filter (the useable clusters)
%>=Q30: % of bases that have a quality score greater than 30 (e.g. high-quality reads)
% aligned: percent of PF reads uniquely aligned to PhiX genome (should be close to the %PhiX spiked in)
Error rate: calculated error rate based on alignment to PhiX
Phasing/Prephasing: percentage of molecules in a cluster that fall behind (phasing) or ahead (prephasing) of the current cycle during sequencing

13. Run statistics - SAV df

14. Considerations for your library
The first 25 bases of a read are used by the instrument
Bases 1-4 used to create cluster ‘map’ – high diversity is critical
Bases 1-12 used for phasing/prephasing calculations
Quality scores and alignment to PhiX start at cycle 26
Phasing/prephasing increases with read length
Cluster images grow with read length and PE turnaround

15. Illumina sequencing Based on reversible terminator chemistry
Sequencing by synthesis (SBS)
All 4 fluorescently labeled bases present