Next-generation sequencing technologies

Transcription

1 Next-generation sequencing technologies

2 NGS applications

3 Illumina sequencing workflow

4 Overview Sequencing by ligation Short-read NGS Sequencing by synthesis Illumina NGS Single-molecule approach Long-read NGS Synthetic approach

5 General principles of short-read NGS Construct a library of fragments Generate clonal template populations Massively parallel DNA sequencing reactions Analyze data

6 General principles of short-read NGS Construct a library of fragments Generate clonal template populations Massively parallel DNA sequencing reactions Analyze data

7 Library preparation Prepares sample nucleic acids for sequencing Fragmentation Generates double-stranded DNA flanked by Illumina adapters Generates the same general template structure, but variables include Insert size Adapter type Index for multiplexing

8 Library preparation: Overview Purified genomic DNA Fragment DNA Repair Ends Fragments < 800bp Add an A to the 3 Ends Ligate Paired-end adapters Blunt end fragments with 5 phosphorylated ends Genomic DNA Library QC Library Amplified DNA with adapters PCR bp fragments Size-select on Gel

9 Library preparation: Fragmentation

10 Library preparation: Fragmentation The size of the target DNA fragments in the final library is a key parameter for NGS library construction. Optimal library size is impacted by 1. the process of cluster generation: Short products amplify more efficiently than longer products. Longer library inserts generate larger, more diffuse clusters than short inserts. 2. the sequencing application: For example, PE for exome sequencing since more than 80% of human exomes are under 200bp.

11 Library preparation: Fragmentation Three approaches are available to fragment nucleic acids: 1. Physical: Acoustic shearing and sonication, main method for genomic DNA 2. Enzymatic: Non-specific endonucleases cocktails or Transposase tagmentation, a greater number of artifactual indels compared with the physical method, reduced sampling handling and preparation time 3. Chemical: Heat and divalent metal cation, reserved for mrna

12 Library preparation: Repair Ends

13 Library preparation: A-tailing

14 Library preparation: A-tailing To facilitate ligation to sequencing adapter To prevent self-ligation between blunt ended template molecules (concatermers), or between adapters (adapter dimers) T P P A A P P A A P P A A P T P P T

15 Library preparation: Adapter ligation

16 Library preparation: Y-shaped adaptors

17 Library preparation: Y-shaped adapters Y-shaped adapters Non Y-shaped adapters

18 Library preparation: Size-select on Gel 600bp area excised 300bp area excised

19 Library preparation: PCR Selectively enrich DNA fragments with adapters on both ends Amplify the amount of DNA in the library

20 Library preparation: PCR

21 Library preparation: QC Library QC by Agilent Bioanalyzer: gives size confirmation and visualizes unwanted products Lower marker 15bp Upper marker 1500bp

22 General principles of short-read NGS Construct a library of fragments Generate clonal template populations Massively parallel DNA sequencing reactions Analyze data

23 Cluster amplification: Flow cells

24 Cluster amplification: Flow cells Adapter-ligated library elements hybridize to complementary oligonucleotides on the surface of a flow cell. Each attached library fragment acted as a seed and is amplified to generate a clonal cluster containing thousands of identical fragments. Ideally, clusters are of similar size and spaced well apart from each other to achieve accurate resolution during imaging. In reality, DNA clusters are randomly distributed across the flow cell with many clusters in close proximity to neighboring clusters, if the sample is overloaded, making it difficult to discern individual clusters from each others and reducing the amount of information generated during the run.

25 Cluster amplification: Patterned flow cells

26 Cluster amplification: Patterned flow cells Patterned flow cell technology provides even cluster spacing and uniform feature size to deliver extremely high cluster densities. Clusters can only form in the nanowells, allowing accurate resolution of clusters during imaging.

27 Cluster amplification

28 Cluster amplification

29 Cluster amplification: Hybridization and extension

30 Cluster amplification: Denaturation

31 Cluster amplification: Anchor the template to the surface

32 Cluster amplification: Bridge amplification

33 Cluster amplification: Bridge amplification

34 Cluster amplification: Denaturation

35 Cluster amplification: Bridge amplification

36 Cluster amplification: Bridge amplification

37 Cluster amplification: P5 Linearization P7 P5

38 Cluster amplification: P5 Linearization

39 Cluster amplification: Blocking

40 Cluster amplification: Read1 sequencing

41 General principles of short-read NGS Construct a library of fragments Generate clonal template populations Massively parallel DNA sequencing reactions Analyze data

42 Sequencing by synthesis

43 Sequencing by synthesis

44 Single read, paired-end and read lengths Program the system to sequence a specific number of bases ( bases) Sequence the strands from both directions to achieve a total of e.g. 600 bases (2 300 bases)

45 Paired-end sequencing Longer read lengths improve 1) the overall length of contiguous sequence that can be assembled, and 2) the certainty of short read alignments. Several next-generation sequencers have offered increases in read length over time. Another improvement has resulted from paired-end sequencing, producing sequence data from both ends of each library fragment. Read pairs can be obtained by one of two mechanisms: 1) paired ends or 2) mate pairs.

46 Paired-end sequencing

47 Paired-end sequencing

48 Paired-end sequencing

49 Paired-end sequencing: P7 linearization

50 Paired-end sequencing

51 Paired-end sequencing Fragment length Advantage (a) paired-end (b) mate-pair < 1000 bp > 1000 bp Higher accuracy of alignments than a single-end read of the same length Providing a scaffold for de novo sequencing by long-range order and orientation

52 Illumina: Summary

53 Illumina platforms: Benchtop sequencers

54 Illumina platforms: Production-scale sequencers

55 Choosing a library type Single read library Unidirectional sequencing Compatible with only single-read flow cells Applications: ChIP-seq, mrna-seq for quantification, low-coverage resequencing

56 Choosing a library type Paired end library Uni or Bidirectional sequencing Compatible with both single-read and paired-end flow cells Applications: the most common library type, de novo assembly, structural variants detection, high-coverage resequencing

57 Choosing a library type Indexed libraries Uni or bidirectional sequencing Allows multiple libraries per lane Single-indexed libraries: adds up to 48 unique 6-base index 1 (i7) se quences to generate up to 48 uniquely tagged libraries. Dual-indexed libraries: adds up to 24 unique 8-base index 1 (i7) sequences and up to 16 unique 8-base index 2 (i5) sequences to generate up to 384 uniquely tagged libraries.

58 Single-indexed sequencing The single-indexed sequencing workflow applies to all Illumina sequencing platforms.

59 Dual-indexed sequencing on a pairedend flow cell Dual-indexed sequencing includes 2 index reads.

60 Dual-indexed adapters

61 Dual-indexed sequencing: Workflow A 7 dark-cycles

62 Dual-indexed sequencing: Workflow A

63 Dual-indexed sequencing: Workflow B

64 Reads and coverage The number of reads for a specific region is denoted depth or coverage