![Page 1: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/1.jpg)
Sequencing Technologies
![Page 2: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/2.jpg)
Illumina Sequencing
2
![Page 3: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/3.jpg)
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 ...
![Page 4: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/4.jpg)
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
![Page 5: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/5.jpg)
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
![Page 6: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/6.jpg)
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
![Page 7: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/7.jpg)
Illumina
UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15
Cluster Generation:Bridge Amplification
● Single-strand flops over 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
![Page 8: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/8.jpg)
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
![Page 9: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/9.jpg)
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 strandReverse strand
![Page 10: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/10.jpg)
Illumina
UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15
Cluster Generation:Bridge Amplification
● Single strands flop over 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
![Page 11: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/11.jpg)
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
![Page 12: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/12.jpg)
Illumina
UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15
Cluster Generation
● dsDNA bridges denatured
● Strands in one of the orientations cleaved and washed away
Illumina: cluster generation
![Page 13: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/13.jpg)
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
![Page 14: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/14.jpg)
TCAGATT
TC
A
G
A
T
T
Illumina: sequencing by synthesis
![Page 15: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/15.jpg)
http://www.slideshare.net/CRS4/chris-jones-crs4-staff-meeting-24032010
Illumina: base calling
![Page 16: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/16.jpg)
16
Number of clusters ~= Number of reads
Number of sequencing cycles ~= Length of reads
![Page 17: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/17.jpg)
Illumina
UC Davis Genome Center | Bioinformatics Core | J Fass HTS 2014-09-15
Cluster Generation:Bridge Amplification
● Single strands flop over 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.
![Page 18: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/18.jpg)
18https://www.illumina.com/systems/sequencing-platforms.html
Illumina: Sequencing Platforms
Benchtop Production-Scale
![Page 19: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/19.jpg)
Benchtop Production-Scale
Benchtop
Production-Scale
![Page 20: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/20.jpg)
20
Other Sequencing Platforms
Pacific Biosciences: http://www.pacb.com/
Oxford Nanopore (MinION): https://nanoporetech.com/
10X Genomics: https://www.10xgenomics.com/
![Page 21: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/21.jpg)
Transcriptomics with long read technologies
21
Advantages Disadvantages
Pacific Biosciences
Iso-Seq protocol for transcripts up to 10Kb, high base calling accuracy
High cost, large machines
Oxford Nanopore
Accurate quantitative data for short transcripts ( < 700bp), portable, high yield
High errors rate affects assembling de novo transcripts, higher amount of cDNA input
10X GenomicsLow cost (integrated with short-read technology), barcoding for accurate isoform detection, low error rates
Extra preparation step (barcode), extra computational step
![Page 22: sequencing technologies mm - GitHub Pages · distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and](https://reader035.vdocuments.site/reader035/viewer/2022062604/5fb4aacd7d982644e533ba11/html5/thumbnails/22.jpg)
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.