User:Jarle Pahr/Sequencing

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[http://maq.sourceforge.net/index.shtml Maq: Mapping and Assembly with Qualities]

Revision as of 17:21, 27 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

Contents

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) AccuracyIntroduced (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

Direct Comparisons of Illumina vs. Roche 454 Sequencing Technologies on the Same Microbial Community DNA Sample

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 2500/1500600 Gb 2 x 100 >80 % higher than Q30
HiSeq 2000/1000300 Gb 2 x 100 >80 % higher than Q30
Genome Analyzer IIx95 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 genome2h+ 100 bp


http://www3.appliedbiosystems.com/cms/groups/applied_markets_marketing/documents/generaldocuments/cms_096460.pdf

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/

Concepts

Targeted sequencing

Targeted "capturing kits" may be used to sequence a subset of genomic DNA. The human exome (as defined by the Consensus CDS (CCDS) project) totals about 38 Mb, covering about 1.22 % of the human genome

(The SureSelect Human All Exon Kit )


Paired-end reads

Haplotypes

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-based techniques

ChIP-sequencing

Sequencing centres

BGI: http://www.genomics.cn/

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 18CAGCGTGCGAGTGATTAT 53.9/60.6 (2)/52.6 (3) 50 Binds upstream of XylS region in pSB-M1g
pJP-1_seq618AGACCACATGGTCCTTCT 57.5° (2)/52.8 ºC(3) 53.9 50 Binds near end of GFPmut3 in pSB-M1g
SeqMG1 AGCAGATCCACATCCTTGAA62.7 (2)/53.7 (3) Binds at nt 5672 of pSB-M1g, upstream of AgeI site. Designed to Macrogen sequencing primer criteria.
pSB-SeqA18TGCAAGAAGCGGATACAG56 / 60.7°C (2)/52.3 ºC (3) 50Binds 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


Maq: Mapping and Assembly with Qualities


Sequencing quality and standards:

http://www.phrap.com/phred/

http://www.bio.net/bionet/mm/autoseq/1999-April/001366.html

http://en.wikipedia.org/wiki/Phred_quality_score

Sequencing projects

File formats

Variant Call Format (VCF):

http://www.1000genomes.org/wiki/Analysis/Variant%20Call%20Format/vcf-variant-call-format-version-41

ABI

FASTq

Bibliography

nar.oxfordjournals.org/content/41/1/e1.full?sid=e66b42ac-a309-47cf-8cd1-94e1229a098e#ref-12


http://online.liebertpub.com/doi/full/10.1089/cmb.2011.0201


Commentary

The state of NGS variant calling - Don't panic: http://blog.goldenhelix.com/?p=1725

http://nxseq.bitesizebio.com/articles/which-way-forward-in-ultra-high-throughput-genomic-sequencing-reference-materials-and-performance-measurements/

Misc

http://nxseq.bitesizebio.com/articles/which-way-forward-in-ultra-high-throughput-genomic-sequencing-reference-materials-and-performance-measurements/

http://genomeinabottle.org/

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