Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

  • #2
    That is very useful, Thanxx.

    Comment


    • #3
      This thread contains some Illumina/Solexa adapter and primer sequences:

      Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)

      Comment


      • #4
        Here's a link to the CHiP-seq Illumina's protocole:


        I don't know if it's relevant in this topic but I thought it could be usefull.

        Comment


        • #5
          Originally posted by huguesparri View Post
          Here's a link to the CHiP-seq Illumina's protocole:


          I don't know if it's relevant in this topic but I thought it could be usefull.
          Excellent! Archived above! Thanks.

          Comment


          • #6
            Recently upgraded on illumina's web site but not obvious to find:

            Comment


            • #7
              Does anybody have an Illumina RNA-Seq prep protocol? Thanks

              Comment


              • #8
                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?

                Comment


                • #9
                  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.



                  Fred

                  Comment


                  • #10
                    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

                    Comment


                    • #11
                      Any information about the way Illumina will achieve 250b reads? like they recently announced.

                      Comment


                      • #12
                        150 bp paired end reads will overlap in the middle of the fragment.

                        Comment


                        • #13
                          Interesting. Thanxx.

                          Comment


                          • #14
                            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!

                            Comment


                            • #15
                              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.
                              Attached Files

                              Comment

                              Latest Articles

                              Collapse

                              • seqadmin
                                Recent Advances in Sequencing Analysis Tools
                                by seqadmin


                                The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...
                                05-06-2024, 07:48 AM
                              • seqadmin
                                Essential Discoveries and Tools in Epitranscriptomics
                                by seqadmin




                                The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
                                04-22-2024, 07:01 AM

                              ad_right_rmr

                              Collapse

                              News

                              Collapse

                              Topics Statistics Last Post
                              Started by seqadmin, 05-14-2024, 07:03 AM
                              0 responses
                              19 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 05-10-2024, 06:35 AM
                              0 responses
                              43 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 05-09-2024, 02:46 PM
                              0 responses
                              53 views
                              0 likes
                              Last Post seqadmin  
                              Started by seqadmin, 05-07-2024, 06:57 AM
                              0 responses
                              42 views
                              0 likes
                              Last Post seqadmin  
                              Working...
                              X