Sequencing Technologies Benchtop Production-Scale Illumina: - - PowerPoint PPT Presentation

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Sequencing Technologies Benchtop Production-Scale Illumina: - - PowerPoint PPT Presentation

Jan 15, 2023 449 likes •684 views

Sequencing Technologies Benchtop Production-Scale Illumina: Sequencing Platforms https://www.illumina.com/systems/sequencing-platforms.html 2 Benchtop Benchtop Production-Scale Production-Scale Nextseq500 HiSeq2500 HiSeq3000/4000

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Sequencing Technologies

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2

https://www.illumina.com/systems/sequencing-platforms.html

Illumina: Sequencing Platforms

Benchtop Production-Scale

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Benchtop Production-Scale

Benchtop Production-Scale

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Illumina: flow cell

Nextseq500 HiSeq2500 HiSeq3000/4000

https://www.illumina.com/company/news-center/multimedia-images.html

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Illumina Sequencing by Synthesis How does it work?

5

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Illumina: flow cell

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

8 "Lanes" (two flow cells per HiSeq) (one-lane FC for MiSeq) Surface of flow cell is coated with a lawn of oligo pairs ...

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Hybridize Fragments & Extend

Adapter Sequence (contains primer)

  • Millions of single

molecules hybridize to the lawn of adapters

  • dsDNA extended by

polymerases

Adapter

Illumina: cluster generation

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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SLIDE 8

Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Denature Double-stranded DNA

  • dsDNA is denatured
  • Original template

fragment washed away

  • Newly synthesized

strand is covalently bound to flow cell

New strand Original strand discard

Original strand Newly synthesized strand

Illumina: cluster generation

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Covalently-Bound, Randomly Dispersed Single Molecules

  • Resulting covalently-

bound DNA fragments are bound to the flow cell surface in a random pattern dsDNA is denatured, original DNA washed away. Newly synthesized strand is covalently bound to flow cell.

Illumina: cluster generation

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • Single-strand flops
  • ver to hybridize to

adjacent adapter, forming a bridge

  • dsDNA synthesized

from primer in hybridized adapter

Illumina: bridge amplification

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • dsDNA bridge now

formed

  • each strand

covalently bound to different adapter

Illumina: bridge amplification

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • dsDNA bridge is

denatured

Illumina: bridge amplification

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

Forward strand Reverse strand

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • Single strands flop
  • ver to hybridize to

adjacent adapters, forming bridges

  • dsDNA synthesized

by polymerases

Illumina: bridge amplification

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • Bridge amplification

cycles repeated many times

Illumina: bridge amplification

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation

  • dsDNA bridges

denatured

  • Strands in one of the
  • rientations cleaved

and washed away

Illumina: cluster generation

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Sequencing By Synthesis

  • Sequencing primer is

hybridized to adapter sequence, starting Sequencing By Synthesis

Sequencing primer http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

Illumina: Prepare for sequencing

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T C A G A T T

T C A G A T T

Illumina: sequencing by synthesis

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http://www.slideshare.net/CRS4/chris-jones-crs4-staff-meeting-24032010

Illumina: base calling

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Number of clusters ~= Number of reads Number of sequencing cycles ~= Length of reads

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Illumina

UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15

Cluster Generation:

Bridge Amplification

  • Single strands flop
  • ver to hybridize to

adjacent adapters, forming bridges

  • dsDNA synthesized

by polymerases

Illumina: paired-end sequencing

http://training.bioinformatics.ucdavis.edu/docs/2014/09/september-2014-workshop/Monday_JF_HTS_lecture.html

dsDNA is denatured, and 3’ ends are de-

  • protected. Template folds over and binds

second oligo on flow cell.

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Other Sequencing Platforms

Pacific Biosciences: http://www.pacb.com/ Oxford Nanopore (MinION): https://nanoporetech.com/ 10X Genomics: https://www.10xgenomics.com/

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Transcriptomics with long read technologies

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Advantages Disadvantages Pacific Biosciences

Iso-Seq protocol for transcripts up to 10Kb, high base calling accuracy High cost, large machines

Oxford Nanopore

Various kits (direct RNA, direct cDNA, cDNA-PCR), short transcripts ( < 700bp), portable, high yield High errors rate affects assembling de novo transcripts, higher amount of cDNA input

10X Genomics

Low cost (integrated with short-read technology), barcoding for accurate isoform detection, low error rates Extra preparation step (barcode), extra computational step

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These materials have been developed by members of the teaching team at the Harvard Chan Bioinformatics Core (HBC). These are open access materials distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.