**Roche GS-FLX (454) Next Generation Sequencing**

![](media/image1.jpeg){width="2.498611111111111in"
height="2.498611111111111in"}

**Introduction to the [GS-FLX ("454")](http://www.454.com/)**

The Roche Genome Sequencer FLX System (GS-FLX or "454") moves DNA
sequencing to a chip based platform dramatically reducing the work and
price required for genome level sequencing projects. This is performed
by simplifying the up front work required to generate and amplify a
genomic library and by massive parallel sequencing in miniature an
entire library simultaneously during a single run on the GS-FLX/454
instrument.

**Applications**

■ *De Novo* Sequencing Supported by Shotgun and Paired-End Applications

■ Comparative Genomics using Whole Genome Sequencing

■ Amplicon Resequencing

■ Transcriptome Analysis

■ Gene Regulation Studies

■ ChIP-Sequencing

**Genome Sequencer FLX System Workflow**

The complete sequencing workflow of the Genome Sequencer FLX System can
be broken down into five steps:

1\. Generation of a single-stranded template DNA library (\~8-16 hours)

2\. Emulsion-based clonal amplification of the library (\~8 hours)

3\. Data generation via sequencing-by-synthesis (9 hours)

4\. Image and Base calling analysis (\~8 hours)

5\. Data analysis using different bioinformatics tools

\- GS-FLX Software

> \- GS Reference Mapper
>
> \- GS De Novo Assembler
>
> \- GS Amplicon Variant Analyzer

\- Third Party Software

**Sample/Library Prep Types**

>  Long Single Reads / Standard Shotgun (required input = 3--5μg, 5μg
> recommended)
>
> \- \~1,000,000 single reads with an average read length of 400 bases

 Paired End Reads (required input = 5μg \@25 ng/μl or above, in TE;
\>10kb)

> \- 3K Long-Tag Paired End Reads. Sequence 100 bases from each end of a
> 3,000 base span on a single sequence read (Figure). Co-assemble GS FLX
> Titanium shotgun reads with 3K Long-Tag Paired Ends reads from
> Standard series runs.
>
>  Sequence Capture (required input = 3--5μg)
>
> \- Roche NimbleGen Sequence Capture enables fast and accurate
> enrichment of thousands of selected genomic regions, either contiguous
> or dispersed (segments of chromosomes, genes, or exons) using a single
> microarray hybridization- based enrichment process.

 Amplicon Sequencing (1-5ng or 10-50ng, see below)

> \- The DNA-sample preparation for Amplicon Sequencing with the GS FLX
> System consists of a simple PCR amplification reaction with special
> Fusion Primers. The Fusion Primer consists of a 20-25 bp
> target-specific sequence (3\' end) and a 19 bp fixed sequence (Primer
> A or Primer B on the 5\' end).
>
> \- If searching for low abundance sequence variants out of a complex
> sample (such as genomic DNA), one should start with 10 -- 50 ng of DNA
> (equivalent to about 3,000 - 15,000 haploid human genomes). If the
> starting material is cloned into a plasmid or is a PCR-generated DNA
> fragment, 1 -- 5 ng is usually sufficient.

### Experimental Design Options

-   Read length options/Sequencing kit formats

    -   GS XLR70 ('Titanium') = \~1,000,000 reads @ \~400bbp

    -   GS LR70 = \~400,000 reads @ \~200-250bp

    -   GS SR70 = \~400,000 reads @ \~100bp

-   PicoTiterPlate gasket formats (A sequencing run can broken into 2,
    4, 8 or 16-regions allowing for a different sample to be run in each
    region)

    -   GS XLR70 'Titanium' Reads per region

        -   2-region format = 450,000-650,000

        -   4-region format = 160,000-250,000

        -   8-region format = 80,000-120,000

        -   16-region format = 25,000-40,000

-   Multiplex Identifiers (MIDs) (Allows for multiple libraries per
    region)

    -   Validated MID barcodes that can be incorporated into libraries

    -   Unique 10-nucleotide sequence

    -   During library preparation, sample-specific MID adaptors
        molecules are blunt-end ligated onto each DNA fragment in a
        sample. The MID-tagged samples can be pooled for simultaneous
        amplification and sequencing in a single region. Used in
        conjunction with the available gaskets, up to 192 samples can be
        sequenced per run. Each MID is recognized by the GS FLX analysis
        software, allowing for automated grouping and analysis of
        MID-containing reads.

-   Targeted region sequencing (Focused region of interest sequencing)

    -   Roche Nimblegen Sequence Capture

    -   Agilent Sequence Capture

**Recommended Reading**

**[General Next-Generation Genome Sequencing]{.mark}**

[**Sequence census methods for functional
genomics**.](http://www.ncbi.nlm.nih.gov/pubmed/18165803) Wold B, Myers
RM (2008) Nat Methods 5(1): 19-21.

[**Approaches to comparative sequence analysis: towards a functional
view of vertebrate
genomes**](http://www.ncbi.nlm.nih.gov/pubmed/18347593). Margulies EH,
Birney E (2008) Nat Rev Genet 9(4): 303-313.

**[Next-generation sequencing transforms today\'s
biology](http://www.ncbi.nlm.nih.gov/pubmed/18165802).** Schuster SC
(2008) Nat Methods 5(1): 16-18.

**[Eukaryotic Whole Genome Sequencing]{.mark}**

[**Identification of ancient remains through genomic
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18426903)  Blow MJ,
Zhang T, Woyke T, Speller CF, Krivoshapkin A, Yang DY, Derevianko A,
Rubin EM.  Genome Research Volume, Ppg.\
7/11/2008\
[**A genome-wide view of the spectrum of spontaneous mutations in
yeast**](http://www.ncbi.nlm.nih.gov/pubmed/18583475)  Lynch M, Sung W,
Morris K, Coffey N, Landry CR, Dopman EB, Dickinson WJ, Okamoto K,
Kulkarni S, Hartl DL, Thomas WK.  PNAS Volume105, Ppg. 9272-9277\
7/8/2008

[**Pyrosequencing as a method for SNP identification in the rhesus
macaque (Macaca
mulatta)**](http://www.ncbi.nlm.nih.gov/pubmed/18510772)  Satkoski JA,
Malhi RS, Kanthaswamy S, Tito RY, Malladi VS, Smith DG  BMC Genomics
Volume9, Ppg. 256\
5/29/2008

[**Global repeat discovery and estimation of genomic copy number in a
large, complex genome using a high-throughput 454 sequence
survey**](http://www.ncbi.nlm.nih.gov/pubmed/17524145)  Kankshita
Swaminathan, Kranthi Varala, and Matthew E. Hudson  BMC Genomics
Volume8, Ppg.\
5/1/2008\
[**The complete genome of an individual by massively parallel DNA
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18421352)   David A.
Wheeler, Maithreyan Srinivasan, Michael Egholm, Yufeng Shen, Lei Chen,
Amy McGuire, Wen He, Yi-Ju Chen, Vinod Makhijani, G. Thomas Roth, Xavier
Gomes, Karrie Tartaro, Faheem Niazi, Cynthia L. Turcotte, Gerard P.
Irzyk, James R. Lupski, Craig Chinault, Xing-zhi Song, Yue Liu, Ye Yuan,
Lynne Nazareth, Xiang Qin, Donna M. Muzny, Marcel Margulies, George M.
Weinstock, Richard A. Gibbs, Jonathan M. Rothberg  Nature Volume452,
Ppg. 872-876\
4/17/2008

[**Paired-End Mapping Reveals Extensive Structural Variation in the
Human Genome**](http://www.ncbi.nlm.nih.gov/pubmed/17901297)   Jan O.
Korbel, Alexander Eckehart Urban, Jason P. Affourtit, Brian Godwin,
Fabian Grubert, Jan Fredrik Simons, Philip M. Kim, Dean Palejev,
Nicholas J. Carriero, Lei Du, Bruce E. Taillon, Zhoutao Chen, Andrea
Tanzer, A. C. Eugenia Saunders, Jianxiang Chi, Fengtang Yang, Nigel P.
Carter, Matthew E. Hurles, Sherman M. Weissman, Timothy T. Harkins, Mark
B. Gerstein, Michael Egholm, and Michael Snyder  Science Volume318, Ppg.
420-426\
9/27/2007

**[Prokaryotic Whole Genome Sequencing]{.mark}**

[**High-throughput sequencing provides insights into genome variation
and evolution in Salmonella
Typhi**](http://www.ncbi.nlm.nih.gov/pubmed/18660809)  Holt KE, Parkhill
J, Mazzoni CJ, Roumagnac P, Weill FX, Goodhead I, Rance R, Baker S,
Maskell DJ, Wain J, Dolecek C, Achtman M, Dougan G.  Nature Genetics
Volume40, Ppg. 987-993\
8/1/2008

[**Genomic content of Bordetella pertussis clinical isolates circulating
in areas of intensive children
vaccination**](http://www.ncbi.nlm.nih.gov/pubmed/18560590)  Bouchez V,
Caro V, Levillain E, Guigon G, Guiso N.  PLoS One Volume3, Ppg.\
7/18/2008

[**The complete genome sequence of a chronic atrophic gastritis
Helicobacter pylori strain: Evolution during disease
progression**](http://www.ncbi.nlm.nih.gov/pubmed/18332421)  Jung D. Oh,
Helene Kling-Backhed, Marios Giannakis, Jian Xu, Robert S. Fulton,
Lucinda A. Fulton, Holland S. Cordum, Chunyan Wang, Glendoria Elliott,
Jennifer Edwards, Elaine R. Mardis, Lars G. Engstrand, and Jeffrey I.
Gordon  PNAS Volume103, Ppg. 9999--10004\
6/7/2008

[**Complete genome of the uncultured Termite Group 1 bacteria in a
single host protist
cell**](http://www.ncbi.nlm.nih.gov/pubmed/18391199)  Yuichi Hongoh,
Vineet K. Sharma, Tulika Prakash, Satoko Noda, Todd D. Taylor, Toshiaki
Kudo, Yoshiyuki Sakaki, Atsushi Toyoda, Masahira Hattori, and Moriya
Ohkuma  PNAS Volume105, Ppg. 5555-5560\
4/8/2008

[**Helicobacter pylori evolution during progression from chronic
atrophic gastritis to gastric cancer and its impact on gastric stem
cells**](http://www.pnas.org/cgi/content/abstract/105/11/4358) 
Giannakis M, Chen SL, Karam SM, Engstrand L, Gordon JI  PNAS Volume105,
Ppg. 4358-4363\
3/18/2008

[**Altered utilization of N-acetyl-D-galactosamine by Escherichia coli
O157:H7 from the 2006 spinach
outbreak**](http://www.ncbi.nlm.nih.gov/pubmed/18156259)  Mukherjee A,
Mammel MK, LeClerc JE, Cebula TA  Journal of Bacteriology Volume190,
Ppg. 1710-1717\
3/1/2008

[**Nanoliter Reactors Improve Multiple Displacement Amplification of
Genomes from Single
Cells**](http://www.ncbi.nlm.nih.gov/pubmed/17892324)  Marcy Y, Ishoey
T, Lasken RS, Stockwell TB, Walenz BP, Halpern AL, Beeson KY, Goldberg
SM, Quake SR  PLoS Genetics Volume3, Ppg.\
9/21/2007

[**The complete genome sequence of Campylobacter jejuni strain 81116
(NCTC11828**](http://www.ncbi.nlm.nih.gov/pubmed/17873037)  Pearson BM,
Gaskin DJ, Segers RP, Wells JM, Nuijten PJ, van Vliet AH  Journal of
Bacteriology Volume, Ppg.\
9/14/2007

[**Assessment of whole genome amplification-induced bias through
high-throughput, massively parallel whole genome
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/16928277)  Robert
Pinard, Alex de Winter, Gary J Sarkis, Mark B Gerstein, Karrie R
Tartaro, Ramona N Plant, Michael Egholm, Jonathan M Rothberg, and John H
Leamon  BMC Genomics Volume7, Ppg.\
8/23/2006

[**A Sanger/pyrosequencing hybrid approach for the generation of
high-quality draft assemblies of marine microbial
genomes**](http://www.ncbi.nlm.nih.gov/pubmed/16840556)  Goldberg SM,
Johnson J, Busam D, Feldblyum T, Ferriera S, Friedman R, Halpern A,
Khouri H, Kravitz SA, Lauro FM, Li K, Rogers YH, Strausberg R, Sutton G,
Tallon L, Thomas T, Venter E, Frazier M, Venter JC  PNAS Volume103, Ppg.
11240-11245\
7/25/2006

**[Ancient DNA]{.mark}**

[**Identification of ancient remains through genomic
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18426903)  Blow MJ,
Zhang T, Woyke T, Speller CF, Krivoshapkin A, Yang DY, Derevianko A,
Rubin EM.  Genome Research Volume, Ppg.\
7/11/2008

[**Intraspecific phylogenetic analysis of Siberian woolly mammoths using
complete mitochondrial
genomes**](http://www.ncbi.nlm.nih.gov/pubmed/18541911)  Gilbert MT,
Drautz DI, Lesk AM, Ho SY, Qi J, Ratan A, Hsu CH, Sher A, Dalén L,
Götherström A, Tomsho LP, Rendulic S, Packard M, Campos PF, Kuznetsova
TV, Shidlovskiy F, Tikhonov A, Willerslev E, Iacumin P, Buigues B,
Ericson PG, Germonpré M, Kosintsev P, Nikolaev V, Nowak-Kemp M, Knight
JR, Irzyk GP, Perbost CS, Fredrikson KM, Harkins TT, Sheridan S, Miller
W, Schuster SC  PNAS Volume105, Ppg. 8327-8332\
6/17/2008

[**From micrograms to picograms: quantitative PCR reduces the material
demands of high-throughput
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18084031)  Meyer M,
Briggs AW, Maricic T, Höber B, Höffner B, Krause J, Weihmann A, Pääbo S,
Hofreiter M  Nucleic Acids Research Volume, Ppg.\
12/15/2007

**[Analysis of one million base pairs of Neanderthal
DNA](http://www.ncbi.nlm.nih.gov/pubmed/17108958/).** Green RE, Krause
J, Ptak SE, Briggs AW, Ronan MT, Simons JF, Du L, Egholm M, Rothberg JM,
Paunovic M, Pääbo S. Nature, 444, (7117), 330-336

11/16/2006

**[ChIP-seq / Methylation / Epigenetics]{.mark}**

[**Nucleosome organization in the Drosophila
genome**](http://www.ncbi.nlm.nih.gov/pubmed/18408708)  Mavrich TN,
Jiang C, Ioshikhes IP, Li X, Venters BJ, Zanton SJ, Tomsho LP, Qi J,
Glaser RL, Schuster SC, Gilmour DS, Albert I, Pugh BF  Nature Volume453,
Ppg. 358-362\
4/18/2008

[**High-Resolution Mapping and Characterization of Open Chromatin across
the
Genome**](http://www.ncbi.nlm.nih.gov/pubmed/18243105?dopt=AbstractPlus) 
Alan P. Boyle, Sean Davis, Hennady P. Shulha, Paul Meltzer, Elliott H.
Margulies, Zhiping Weng, Terrence S. Furey, and Gregory E. Crawford 
Cell

Volume132, Ppg. 311--322\
1/25/2008

[**Identification of Novel High-Frequency DNA Methylation Changes in
Breast Cancer**](http://www.ncbi.nlm.nih.gov/pubmed/18091988)  Jared M.
Ordway, Muhammad A. Budiman, Yulia Korshunova, Rebecca K. Maloney,
Joseph A. Bedell, Robert W. Citek, Blaire Bacher, Seth Peterson, Tracy
Rohlfing, Jacqueline Hall, Robert Brown, Nathan Lakey, Rebecca W.
Doerge, Robert A. Martienssen, Jorge Leon, John D. McPherson, Jeffrey A.
Jeddeloh  PLoS ONE Volume12, Ppg.\
12/1/2007

[**Flexibility and constraint in the nucleosome core landscape of
Caenorhabditis elegans
chromatin**](http://www.genome.org/cgi/content/abstract/gr.5560806v1) 
Steven M. Johnson, Frederick J. Tan, Heather L. McCullough, Daniel P.
Riordan, and Andrew Z. Fire  Genome Research Volume16, Ppg. 1505 - 1516\
12/1/2006

**[Sequence Capture / Target Region Sequencing]{.mark}**

[**Direct resequencing of the complete ERBB2 coding sequence reveals an
absence of activating mutations in ERBB2 amplified breast
cancer**](http://www.ncbi.nlm.nih.gov/pubmed/18418848)  Zito CI, Riches
D, Kolmakova J, Simons J, Egholm M, Stern DF.  Genes Chromosomes Cancer
Volume47, Ppg. 633-638\
7/1/2008

[**Direct selection of human genomic loci by microarray
hybridization**](http://www.ncbi.nlm.nih.gov/pubmed/17934467)  Albert
TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA,
Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA  Nature
Methods Volume4, Ppg. 903-905\
11/1/2007

**[Transcriptome / Small RNA Sequencing]{.mark}**

[**Detection of large numbers of novel sequences in the
metatranscriptomes of complex marine microbial
communities**](http://www.ncbi.nlm.nih.gov/pubmed/18725995)  Gilbert JA,
Field D, Huang Y, Edwards R, Li W, Gilna P, Joint I.  PLoS ONE Volume3,
Ppg. e3042\
8/22/2008

[**Development of a DNA barcode tagging method for monitoring dynamic
changes in gene expression by using an ultra high-throughput
sequencer**](http://www.ncbi.nlm.nih.gov/pubmed/18611171)   Maeda N,
Nishiyori H, Nakamura M, Kawazu C, Murata M, Sano H, Hayashida K, Fukuda
S, Tagami M, Hasegawa A, Murakami K, Schroder K, Irvine K, Hume D,
Hayashizaki Y, Carninci P, Suzuki H.  Biotechniques Volume45, Ppg.
95-97\
7/1/2008

[**Rapid transcriptome characterization for a non-model organism using
454 pyrosequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18266620)  J.
C. Vera, C.W. Wheat, H.W. Fescemyer, M.J. Frilander, D.L. Crawford, I.
Hanski, J.H. Marden  Molecular Ecology Volume17, Ppg. 1636-1647\
2/5/2008

[**SNP discovery via 454 transcriptome
sequencing**](http://www.ncbi.nlm.nih.gov/pubmed/17662031)  Barbazuk WB,
Emrich SJ, Chen HD, Li L, Schnable PS  The Plant Journal Volume51, Ppg.
910-918\
7/1/2007

[**Early origins and evolution of microRNAs and Piwi-interacting RNAs in
animals.**](http://www.ncbi.nlm.nih.gov/pubmed/18830242)  Grimson A,
Srivastava M, Fahey B, Woodcroft BJ, Chiang HR, King N, Degnan BM,
Rokhsar DS, Bartel DP.  Nature VolumeePub, Ppg.\
10/1/2008

**[Metagenomic and Microbial Diversity]{.mark}**

[**Comparative analysis of human gut microbiota by barcoded
pyrosequencing**](http://www.ncbi.nlm.nih.gov/pubmed/18665274) 
Andersson AF, Lindberg M, Jakobsson H, Bäckhed F, Nyrén P, Engstrand L. 
PLoS One Volume3, Ppg. e2836\
7/30/2008

[**Functional metagenomic profiling of nine
biomes**](http://www.ncbi.nlm.nih.gov/pubmed/18337718)  Dinsdale EA,
Edwards RA, Hall D, Angly F, Breitbart M, Brulc JM, Furlan M, Desnues C,
Haynes M, Li L, McDaniel L, Moran MA, Nelson KE, Nilsson C, Olson R,
Paul J, Brito BR, Ruan Y, Swan BK, Stevens R, Valentine DL, Thurber RV,
Wegley L, White BA, Rohwer F  Nature Volume452, Ppg. 629-632\
4/3/2008

[**Microbial ecology of four coral atolls in the northern line
islands**](http://www.ncbi.nlm.nih.gov/pubmed/18301735)  Dinsdale EA,
Pantos O, Smriga S, Edwards RA, Angly F, Wegley L, Hatay M, Hall D,
Brown E, Haynes M, Krause L, Sala E, Sandin SA, Thurber RV, Willis BL,
Azam F, Knowlton N, Rohwer F  PLoS ONE Volume3, Ppg.\
2/27/2008

[**Bacterial carbon processing by generalist species in the coastal
ocean**](http://www.ncbi.nlm.nih.gov/pubmed/18223640)  Mou X, Sun S,
Edwards RA, Hodson RE, Moran MA  Nature Volume451, Ppg. 708-711\
2/7/2008

**Contact Information**

Questions relating to the HGAC including pricing should be directed to
Marc Domanus, Laboratory Research Manager as described below.

Email: <sequencing@igsb.anl.gov>

IGSB High-throughput Genome Analysis Core (HGAC)

[Argonne National Laboratory](http://www.anl.gov/Visiting/anlemap.html)

9700 South Cass Avenue

Building 202 A353

Argonne, IL 60439-4803

Phone: (630) 252-3993

Fax: (630) 252-6333

LINKS

> Platforms
>
> [Roche GS FLX (454) Sequencing]{.underline}
>
> [Illumina Solexa Next Generation Sequencing]{.underline}
>
> [Microarray Processing]{.underline}
>
> [Sample Submission Guidelines]{.underline}