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Old 12-03-2007, 08:13 PM   #1
ECO
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Default Tech Summary: Illumina's Solexa Sequencing Technology



Illumina's $600 million acquisition of Solexa in November 2006 gave the company a head start in the next generation sequencing market.

Here I present a brief overview of Solexa's sequencing-by-synthesis chemistry. The sample prep methods used differ slightly from that used in ABI's SOLiD system, but the basic goals are the same: generate large numbers of unique "polonies" (polymerase generated colonies) that can be simultaneously sequenced. These parallel reactions occur on the surface of a "flow cell" (basically a water-tight microscope slide) which provides a large surface area for many thousands of parallel chemical reactions.

Step 1: Sample Preparation


The DNA sample of interest is sheared to appropriate size (average ~800bp) using a compressed air device known as a nebulizer. The ends of the DNA are polished, and two unique adapters are ligated to the fragments. Ligated fragments of the size range of 150-200bp are isolated via gel extraction and amplified using limited cycles of PCR.

Complete detailed protocols for DNA and small RNA library preparation can be found in the documents provided in the attachments to this post. ("dna_libe_prep.pdf" and "rna_libe_small_prep.pdf", respectively). This process is a fairly straightforward multi-step molecular biology process, however there are many pitfalls that can result in skewed results downstream.

Steps 2-6: Cluster Generation by Bridge Amplification

In contrast to the 454 and ABI methods which use a bead-based emulsion PCR to generate "polonies", Illumina utilizes a unique "bridged" amplification reaction that occurs on the surface of the flow cell.

The flow cell surface is coated with single stranded oligonucleotides that correspond to the sequences of the adapters ligated during the sample preparation stage. Single-stranded, adapter-ligated fragments are bound to the surface of the flow cell exposed to reagents for polyermase-based extension. Priming occurs as the free/distal end of a ligated fragment "bridges" to a complementary oligo on the surface.

Repeated denaturation and extension results in localized amplification of single molecules in millions of unique locations across the flow cell surface. This process occurs in what is referred to as Illumina's "cluster station", an automated flow cell processor.



Steps 7-12: Sequencing by Synthesis

A flow cell containing millions of unique clusters is now loaded into the 1G sequencer for automated cycles of extension and imaging.

The first cycle of sequencing consists first of the incorporation of a single fluorescent nucleotide, followed by high resolution imaging of the entire flow cell. These images represent the data collected for the first base. Any signal above background identifies the physical location of a cluster (or polony), and the fluorescent emission identifies which of the four bases was incorporated at that position.

This cycle is repeated, one base at a time, generating a series of images each representing a single base extension at a specific cluster. Base calls are derived with an algorithm that identifies the emission color over time. At this time reports of useful Illumina reads range from 26-50 bases.




The use of physical location to identify unique reads is a critical concept for all next generation sequencing systems. The density of the reads and the ability to image them without interfering noise is vital to the throughput of a given instrument. Each platform has its own unique issues that determine this number, 454 is limited by the number of wells in their PicoTiterPlate, Illumina is limited by fragment length that can effectively "bridge", and all providers are limited by flow cell real estate.

Hopefully that serves as a brief introduction to the technology! If I have made any errors or omissions, please feel free to correct me by posting here!
Attached Files
File Type: pdf dna_libe_prep.pdf (153.3 KB, 3601 views)
File Type: pdf rna_libe_small_prep.pdf (125.2 KB, 1930 views)
File Type: pdf PreparingSamplesforDigitalGeneExpression-TagProfilingwithNlaIII.pdf (151.7 KB, 1235 views)
File Type: pdf PreparingSamplesforDigitalGeneExpression-TagProfilingwithDpnII.pdf (150.5 KB, 882 views)
File Type: pdf 11257047_ChIP_Sample_Prep.pdf (116.2 KB, 1353 views)
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Old 04-04-2008, 04:46 AM   #2
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That is very useful, Thanxx.
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Old 04-07-2008, 03:30 PM   #3
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This thread contains some Illumina/Solexa adapter and primer sequences:

http://seqanswers.com/forums/showthread.php?t=198
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Old 05-09-2008, 12:51 AM   #4
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Here's a link to the CHiP-seq Illumina's protocole:
http://www.rockefeller.edu/genomics/...ample_Prep.pdf

I don't know if it's relevant in this topic but I thought it could be usefull.
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Old 05-09-2008, 06:38 AM   #5
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Quote:
Originally Posted by huguesparri View Post
Here's a link to the CHiP-seq Illumina's protocole:
http://www.rockefeller.edu/genomics/...ample_Prep.pdf

I don't know if it's relevant in this topic but I thought it could be usefull.
Excellent! Archived above! Thanks.
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Old 05-23-2008, 04:04 AM   #6
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Recently upgraded on illumina's web site but not obvious to find:
http://www.illumina.com/seminars/arc..._GA_Update.pdf
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Old 09-21-2008, 12:16 PM   #7
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Does anybody have an Illumina RNA-Seq prep protocol? Thanks
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Old 10-16-2008, 08:05 AM   #8
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Default Fluorophore removal

How does Illumina remove the fluorophore? They can't use AgNO3 (like SOLiD ) because of no Thio backbone. Are they using the Columbia IP that Intelligent BioSystems is using? http://www.intelligentbiosystems.com...20mod%201.html.

Any thoughts here?
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Old 02-05-2009, 06:09 AM   #9
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Hi

Illumina Presents Development Roadmap for Scaling its Genome Analyzer

They plan to sequence DNA fragments of up to 250 base pairs generating a 25x coverage human geneome on a single flow cell.

http://investor.illumina.com/phoenix...407&highlight=

Fred
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Old 02-08-2009, 01:40 PM   #10
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This site is useful as well for a collection of library protocols from Illumina: http://keck.med.yale.edu/microarrays...protocols.html

It gives pdf library protocols for the following:

Preparing Samples for Paired-End Sequencing

Preparing Samples for Sequencing Genomic DNA

Preparing Samples for Digital Gene Expression-Tag Profiling with NlaIII

Preparing Samples for Digital Gene Expression-Tag Profiling with DpnII

Preparing Samples for Analysis of Small RNA

Preparing Samples for ChIP Sequencing of DNA

Preparing Samples for Whole Transcriptome Profiling

Preparing Samples for Multiplexed Paired End Sequencing

Preparing Samples for Paired-End Sequencing
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Old 02-08-2009, 01:59 PM   #11
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Any information about the way Illumina will achieve 250b reads? like they recently announced.
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Old 02-08-2009, 02:06 PM   #12
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150 bp paired end reads will overlap in the middle of the fragment.
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Old 02-08-2009, 02:15 PM   #13
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Interesting. Thanxx.
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Old 03-01-2009, 10:08 PM   #14
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hello everybody! This is my first post here,I need the composition of the washingbuffer、binding buffer、 bufferCand D used in the illumina gene expression kit with NlaIII.If anyone could help me ,Iwill be very helpful!
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Old 05-26-2009, 04:38 PM   #15
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Default adapter illustration diagram

I didn't really understand the Illumina library prep and sequencing procedure until I played around with aligning the actual sequences. This resulted in a diagram of the ligation/PCR/hybridization/sequencing process for both single read and paired end reads using the publicly available adapter, primer, and flow cell oligo sequences. Hope it is useful to others, and let me know if you find errors. Thanks to kmcarr for posting your diagram of part of the single read process earlier, it inspired me to work it out for myself. You can find all the sequences I used in the 2008 nature paper, doi 10.1038/nature07517.
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Old 06-25-2009, 08:45 AM   #16
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I have a question about the adapter sequences; for one of the adapter sequences you have a single base difference between the SR adapter and the PE adapter. In the official communication from Illumina that lists all their sequences there is no difference in the unphosphorylated adapter sequence between SR and PE adapters they are both 33 bases long. Where did you get the sequences from (as it's not the first time I've heard about the single base difference but there's definitely none in the sequences that I have).
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Old 09-29-2009, 05:30 PM   #17
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I am a little confused about this.

In fig 6, the DNA is attached to flow-cell by both red and purple adapters in the same cluster. However, in fig 7, it is attached only by red adapters.

What causes it to happen?

Both adapters would mean the DNA strands in clusters are not identical, but half of them are complimentary to the other half.
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Old 09-30-2009, 05:50 AM   #18
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When beginning the sequencing, first a cleaving step is carried out,
removing the sequences bound with adapter2 to the cell-surface.

For paired-end sequencing, after having reached the desired cycle-count the synthesized strand is removed, another step of bridge amplification is carried out, followed by a cleaving of sequences bound with adapter1 to the cell surface. Thus leaving the `other'/reverse strand bound to the flow cell for sequencing...

Anything else?

Last edited by Jonathan; 09-30-2009 at 05:55 AM.
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Old 11-10-2009, 11:48 AM   #19
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Default why PE1 and SE1 adapter sequece has 1 base different?

I found that they should be identical. The attached diagram is wrong.

compare diagram 1 and 2, adaptor sequence is changed!!!


Quote:
Originally Posted by greigite View Post
I didn't really understand the Illumina library prep and sequencing procedure until I played around with aligning the actual sequences. This resulted in a diagram of the ligation/PCR/hybridization/sequencing process for both single read and paired end reads using the publicly available adapter, primer, and flow cell oligo sequences. Hope it is useful to others, and let me know if you find errors. Thanks to kmcarr for posting your diagram of part of the single read process earlier, it inspired me to work it out for myself. You can find all the sequences I used in the 2008 nature paper, doi 10.1038/nature07517.
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Old 11-10-2009, 01:10 PM   #20
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Post Software choices?

Hi there,

I have a question about the software I can use to analyze the Illumina sequencing data. I'm doing targeted resequencing of long PCR fragments to find some EMS mutations. I'm waiting for my run to finish right now and meanwhile I'm looking for a suitable software. Any suggestion would be of great help to me. Thank you.
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