Introduction to Illumina Sequencing

Day 1, Video 2
• Overview of Next-gen sequencing

• Introduction to Illumina sequencing

• Multiplexing

• Sequencing run statistics

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)
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
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’
Illumina sequencing
• SBS chemistry video
• http://www.illumina.com/technology/next-generation-seq
uencing/sequencing-
technology.html
Clustering, the first step to sequencing
Sequencing by Synthesis overview
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!
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+)
Multiplexing – single index read
Multiplexing – dual index reads
• hf
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
Run statistics - SAV
• df
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
Illumina sequencing
• Based on reversible terminator chemistry
• Sequencing by synthesis (SBS)
• All 4 fluorescently labeled bases present