User:Jarle Pahr/Sequencing

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

Technologies

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

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 2500/1500	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

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/

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

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.phrap.com/phred/

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