User:Jarle Pahr/Sequencing: Difference between revisions
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http://www.ki.se/kiseq/KIGene%20troubleshooting.pdf | http://www.ki.se/kiseq/KIGene%20troubleshooting.pdf | ||
Nature focus issue - sequencing technology: http://www.nature.com/nbt/journal/v30/n11/index.html | |||
=Technologies= | =Technologies= | ||
Revision as of 12:19, 25 March 2013
http://nucleicacids.bitesizebio.com/articles/how-to-get-great-dna-sequencing-results/
http://barricklab.org/twiki/bin/view/Lab/ProceduresPrimerDesign
http://www.ki.se/kiseq/KIGene%20troubleshooting.pdf
Nature focus issue - sequencing technology: http://www.nature.com/nbt/journal/v30/n11/index.html
Technologies
For a comparison of next-generation sequencing methods, see http://en.wikipedia.org/wiki/Dna_sequencing#Next-generation_methods
Sanger sequencing (chain termination method)
Pyrosequencing ("454 sequencing")
Pyrosequencing is a "sequence by synthesis" method developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of Technology, Stockholm. Sequences are determined by observation of light emission upon addition of a nucleotide complementary to the first unpaired nucleotide of the template.
Quote from Wikipedia:Pyrosequencing:
"ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin."
Sequencing proceeds as follows:
- Addition of one of the four dNTPs (dATPαS is substituted for ATP, as the former is not a substrate for luciferase). If the dNTP is complementary, DNA polyerase incorporates the nucleotide, releasing pyrophosphate (PPi).
- ATP sulfurylase catalyzes reaction of PPi and adenosine 5' phosphosulfate to create ATP
- ATP fuels luciferase-catalyzed conversion of luciferin to oxyluceferin, generating visible light.
- Unincorporated nucleotides and ATP are degraded by apyrase.
454 sequencing performs massively parallel pyrosequencing. Library DNA containing adapter sequences are adsorbed to DNA-capturing beads. The DNA bound to each bead is then amplified by emulsion-PCR, in which the beads with bound DNA are mixed with PCR reagents and emulsion oil to create a water-in-oil emulsion containing many "microreactors" consisting of beads sorrounded by water. Following PCR amplification, the DNA-binding beads are isolated and deposited into the wells of a microtiter plate. Beads with pyrosequencing enzymes are then added to the plate. Finally, the pyrosequencing is performed, processing the plate in a sequencing machine. 400 000+ DNA fragments/beads can be processed per plate.
Using "multiplex identifiers", different genomic libraries can be bar-coded, facilitating sequencing of several libraries in the same sequencing run.
Platforms:
Platform | Throughput (bases/run) | Time per run | Average (a)/mode (m) read length (nt) | Accuracy | Introduced (year) |
---|---|---|---|---|---|
GS FLX+ | 700 Mbp | 23h | Up to 1000 | 700 bp (m) | |
GS Junior | 35Mbp | 12 h | 400 | 400 bp (a) at Phred20/read |
GS FLX:
References:
Introductory paper, 454 sequencing: http://www.ncbi.nlm.nih.gov/pubmed/16056220?dopt=Abstract&holding=npg
http://www.wellcome.ac.uk/Education-resources/Education-and-learning/animations/dna/wtx056046.htm
The development and impact of 454 sequencing
Overview of 454 sequencing: http://classes.soe.ucsc.edu/bme215/Spring09/PPT/BME%20215-5.pdf
Illumina (Solexa) sequencing
http://www.illumina.com/technology/sequencing_technology.ilmn
Platform | Throughput (bases/run) (maximum) | Time per run | Read length (nt) | Accuracy | Features | Introduced (year) |
---|---|---|---|---|---|---|
MiSeq Personal Sequencer | Up to 8.5 gbp | 4 - 48 h | 250 | >70% bases higher than Q30 at read length 2 x 300 bp | ||
HiSeq 2500/1500 | 600 Gb | 2 x 100 | >80 % higher than Q30 | |||
HiSeq 2000/1000 | 300 Gb | 2 x 100 | >80 % higher than Q30 | |||
Genome Analyzer IIx | 95 Gb | 2 x 150 | >80 % higher than Q30 |
MiSeq datasheet: http://www.illumina.com/documents/products/datasheets/datasheet_miseq.pdf
Side by side comparison of Illumina sequencers: http://www.illumina.com/systems/sequencing.ilmn
Illumina - an introduction to NGS: http://www.illumina.com/Documents/products/Illumina_Sequencing_Introduction.pdf
Ion semiconductor sequencing
Ion Torrent: http://www.invitrogen.com/site/us/en/home/brands/Ion-Torrent.html?cid=fl-iontorrent Platforms:
Platform | Throughput (bases/run) | Time per run | Typical read length | Accuracy | Introduced (year) |
---|---|---|---|---|---|
Ion PGM sequencer | 10 Mb to 1Gb | 90 min+ | 35-400 bp | ||
Ion Proton sequencer | 1 human genome | 2h+ | 100 bp |
Nanopore sequencing
Oxford Nanopore: http://www.nanoporetech.com/
Single molecule real time sequencing (Pacific Biosciences)
Microscopical wells on a chip (zero-mode waveguides) each contain a single DNA polymerase enzyme bound to the bottom of the well, which accept a single DNA molecule as template. Fluorescent labelled dNTPs are used for DNA synthesis. Upon incorporation of a dNTP, the fluorescence tag is cleaved from the nucleotide and diffuses from the observation area within the ZMW. The sequence is determined optically by observing incorporation events.
http://www.pacificbiosciences.com/
SOLiD sequencing (Applied Biosystems)
DNA nanoball sequencing
http://www.completegenomics.com/services/technology/
Sequencing services
Service | Sample specification | Primer specification | Ship to | Link |
---|---|---|---|---|
GATC LightRun | Add 5 uL DNA (80-100 ng/uL plasmid or 20-80 ng/uL purified PCR product) + 5 uL 5uM (5 pmol/uL) primer to the same tube | Tm 52-58 C, 17-19 bp, (8-9 G+C for 18-mer) G or C at 3' end (max 3 Gs or Cs), maximum 4bp run. | GATC Biotech AG. European Custom Sequencing Centre. Gotrfied-Hagen-Strasse 20. 51105 Köln. | http://www.gatc-biotech.com/en/lp4/new-lightrun-sequencing.html |
Macrogen Single-pass | Add 20 uL DNA (100 ng/uL plasmid or 50 ng/uL purified PCR product) to one tube. Add 20µl primer (10 pmol/uL) to a separate tube. | 18-25 bp, 40-60 % GC, Tm 55-60 | Macrogen Europe,
IWO, Kamer IA3-195, Meibergdreef 39,1105 AZ Amsterdam Zuid-oost. Netherlands. Attention: J.S .Park. |
http://dna.macrogen.com/eng/support/seq/seq_submission.jsp |
Sequencing centres
New York Genome Center: http://nygenome.org/
Primers
Custom primers
Name | Length (bp) | Sequence | Tm (C) [calculated] | Tm (C) [Analytical] | GC (% / bp) | Comment |
---|---|---|---|---|---|---|
pJP-1_seq5 | 18 | CAGCGTGCGAGTGATTAT | 53.9/60.6 (2)/52.6 (3) | 50 | Binds upstream of XylS region in pSB-M1g | |
pJP-1_seq6 | 18 | AGACCACATGGTCCTTCT | 57.5° (2)/52.8 ºC(3) | 53.9 | 50 | Binds near end of GFPmut3 in pSB-M1g |
SeqMG1 | AGCAGATCCACATCCTTGAA | 62.7 (2)/53.7 (3) | Binds at nt 5672 of pSB-M1g, upstream of AgeI site. Designed to Macrogen sequencing primer criteria. | |||
pSB-SeqA | 18 | TGCAAGAAGCGGATACAG | 56 / 60.7°C (2)/52.3 ºC (3) | 50 | Binds at nt 7729 of pSB-M1g, upstream of Pm promoter and PciI site. |
Universal primers
http://www.generi-biotech.com/sequencing-universal-seguencing-primers/ http://www.synthesisgene.com/tools/Universal-Primers.pdf http://www.genewiz.com/public/universalprimers.aspx https://secure.eurogentec.com/product/research-universal-primers.html
Tm calculations:
1: CloneManager
2: Thermo Scientific
3: IDT Oligoanalyzer
A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers
http://www.biomedcentral.com/1471-2164/13/341
Software
Chromatogram viwers: http://www.dnaseq.co.uk/chrom_view.html
CodonCode aligner: http://www.codoncode.com/aligner/
BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html
FinchTV: http://www.geospiza.com/Products/finchtv.shtml
About SCF (sequence chromatogram format) files: http://staden.sourceforge.net/manual/formats_unix_2.html
https://wiki.nci.nih.gov/display/TCGA/Sequence+trace+files
http://code.google.com/p/seqtrace/
http://www.phrap.com/background.htm
http://en.wikipedia.org/wiki/Phrap
http://www.ncbi.nlm.nih.gov/books/NBK47537/
http://www.bio.net/bionet/mm/autoseq/1999-April/001368.html
Sequencing quality and standards:
http://www.bio.net/bionet/mm/autoseq/1999-April/001366.html
http://en.wikipedia.org/wiki/Phred_quality_score
Bibliography
nar.oxfordjournals.org/content/41/1/e1.full?sid=e66b42ac-a309-47cf-8cd1-94e1229a098e#ref-12