This document provides an overview of Illumina sequencing, including: - Illumina sequencing uses a sequencing by synthesis (SBS) approach with reversible terminator chemistry. All four fluorescently labeled bases are present in each sequencing cycle. - Key steps include library construction, cluster generation, bridge amplification on the flow cell, and single-base sequencing imaging. - Multiplexing allows indexing of multiple samples by attaching barcodes during library preparation. This enables pooled sequencing of many samples. - Run statistics like number of reads, percentage of high-quality bases, and alignment rates provide information about run quality and performance.

1. Illumina Sequencing
•
• Scientific Zone

2. Introduction to Illumina Sequencing
• Overview of Next-gen sequencing
• Introduction to Illumina sequencing
• Multiplexing
• Sequencing run statistics

3. 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)

4. 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

5. 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’

6. Illumina sequencing
• SBS chemistry video
• http://www.illumina.com/technology/next-generation-
sequencing/sequencing-technology.html

7. Clustering, the first step to sequencing

8. Sequencing by Synthesis overview

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

10. Anatomy of a library
• P5 and P7 ends of adapters bind to flow cell
• DNA insert typically ranges 200-600 bp (<1kb)
• Different methods of indexing
• Inline (part of the insert) – any level of multiplexing
• Single index read (≤96)
• Dual index reads (384+)
5 This content is for research use only, not for diagnostic purposes
The aim of the sample prep step is to obtain nucleic
acid fragments with adapters attached on both ends
Dual Index Library shown

11. Multiplexing – single index read
38 FOR RESEARCH USE ONLY
Read 2 Seq Primer
(HP7)
Read 1 Seq Primer
(HP6)
Index Seq Primer
(HP8)
1
2
3
Multiplex Sequencing Utilizes 3 Sequencing Reads
Paired End
Turnaround
Single Index Sequencing Utilizes 3 Sequencing Reads
Sequencing with Paired Ends

12. Multiplexing – dual index reads
• hf
40 FOR RESEARCH USE ONLY
1 2 3
Paired End
Turnaround
4
Dual Index Sequencing Utilizes 4 Sequencing Reads
Sequencing Paired End Libraries with Dual Index Read

13. 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

14. Run statistics - SAV
• df

15. 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

16. Illumina sequencing
• Based on reversible terminator chemistry
• Sequencing by synthesis (SBS)
• All 4 fluorescently labeled bases present
24 FOR RESEARCH USE ONLY
• All 4 labeled nucleotides in 1 reaction
• Higher accuracy
• No problems with homopolymer repeats
• Incorporation
• Detection
• Deblock
• Fluor Removal
Next Cycle
Reversible Terminator Chemistry