NextGen Sequencing Technologies Sequencing overview
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1 Outline Conventional NextGen High-throughput sequencing (Next-Gen sequencing) technologies. Illumina sequencing in detail. Quality control. Sequence coverage. Multiplexing. FASTQ files. Shendure and Ji (2008) NextGen Sequencing Technologies Sequencing overview RNA or DNA isolation from cells or tissue Library preparation (often involves kits) Size selection and clean up Quantification (often by qpcr) Pooling of indexed libraries (if applicable) Sequencing (typically by a core lab or company) MacLean et al (2009) Michael L. Metzker (2010) Illumina sequencing 1800Gb 6B 2x150 Slides used with permission from Illumina. Increasing System Price & Output 15Gb 25M 2x Gb 400M 2x Gb 4B 2x125 HiSeq Ten NextSeq HiSeq 2500 MiSeq Decreasing Price Per Gb 6 1
2 Library Prep is Critical for Successful Sequencing What is a Flow Cell? Cluster generation occurs on a flow cell A flow cell is a thick glass slide with channels or lanes Dual Index Library shown The aim of the sample prep step is to obtain nucleic acid fragments with adapters attached on both ends Each lane is randomly coated with a lawn of oligos that are complementary to library adapters 7 8 Denature Double-ed DNA Double-ed molecule is denatured Original template Newly synthesized Single-ed molecule flips over and forms a bridge by hybridizing to adjacent, complementary primer Original template washed away discard Hybridized primer is extended by polymerases Newly synthesized is covalently attached to flow cell surface 9 10 Denature Double-ed Bridge Double-ed bridge is formed Double-ed bridge is denatured Result: Two copies of covalently bound single-ed templates
3 Single-ed molecules flip over to hybridize to adjacent primers Bridge amplification cycle repeated until multiple bridges are formed Hybridized primer is extended by polymerase 13 Linearization dsdna bridges are denatured 15 Blocking Free 3 ends are blocked to prevent unwanted DNA priming Reverse Strand Cleavage Reverse s cleaved and washed away, leaving a cluster with forward s only 16 Read 1 Primer Hybridization Sequencing primer is hybridized to adapter sequence Sequencing primer 18 3
4 Sequencing By Synthesis Sequenced is stripped off Blocked 3 -ends Sequenced 3 -ends of template s and lawn primers are unblocked Add 4 Fl-NTP s + Polymerase Incorporated FI- NTP imaged Terminator & fluorescent dye cleaved from FI-NTP Single-ed template loops over to form a bridge by hybridizing with a lawn primer 3 -ends of lawn primer is extended Bridge formation Double ed DNA 3 extension Bridges are linearized and the original forward template is cleaved Free 3 ends of the reverse template and lawn primers are blocked to prevent unwanted DNA priming Blocked 3 -ends Sequencing primer Original forward Sequencing primer is hybridized to adapter sequence Reverse template
5 Sequencing By Synthesis 2 nd Read Data Quality Add 4 Fl-NTP s + Polymerase Incorporated FI- NTP imaged Terminator & fluorescent dye cleaved from FI-NTP Q bp Q bp Quality control Coverage FastQC: a quality control tool for high-throughput sequencing data Alignment 3 2 reference 28 Indexing pooling samples DNA/RNA samples unique sequence barcode create library & add index increase throughput indexed libraries maximize capacity TAAGGCGAGTA.. CGTACTAGTGG.. pool informatic de-multiplex AGGCAGAAAGT.. TCCTGAGCTGC.. GGACTCCTGTG.. TAGGCATGTAG.. FASTQ CTCTCTACGCA.. CAGAGAGGAGT.. GCTACGCTGTG.. CGAGGCTGTAG.. AAGAGGCAGCA.. GTAGAGGAAGT
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