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-sequencing/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