Sekans Teknolojilerinin Gelişmi

Sekanslama süreci Örnek hazırlama-hedef genomun küçük parçalara ayrılması Fiziksel sekanslama- her parçadaki bazların sırayla tayini Geri birleştirme- üst üste gelen sekansların hizalanması

1. Nesil sekanslama (1st generation) Sanger(in 1975), Maxam and Gilbert (in 1977) dideoxy nucleotides,floresan ile işaretlenmiş Kapiler elektroforez Videos

1st generation sekanslama Sanger-zincir sonlandırma metodu (chain termination method)

SANGER SEQ http://www.youtube.com/watch?v=6ldtdWjDwes &feature=related http://www.youtube.com/watch?v=bEFLBf5WEt c&NR=1&feature=endscreen

Sekans kromatogramlarının analizi Sekans sonuçlarının iyi değerlendirilmesi için kromatogramların indirilmesi ve detaylı analiz edilmesi gerekir. Sadece cihazdan alınan text dosyası ile işlem yapılıyorsa, güvenilir sonuçlar elde edilemeyebilir. Automated DNA Sequencers generate a four-color chromatogram showing the results of the sequencing run, as well as a computer program's best guess at interpreting that data - a text file of sequence data. That computer program, however, does make mistakes and you need to manually double-check the interpretation of the primary data. Predictable errors occur near the beginning and again at the end of any sequencing run. Other errors can crop up in the middle, invalidating individual base calls or entire swaths of data.

Kısa sürede kromatogram okuma.

1. Sekansın ne kadar temiz olduğu ile ilgili fikir edinin Genel olarak nükleotid pikleri ne kadar temiz? Eşit aralıklarla ayrılmış ve her biri farklı renklerde pik görüntüsü olmalı. «Gürültü» (baseline) pikleri olabilir ancak iyi bir şablon DNA ve iyi primerler ile minimum seviyeye indirilebilir.

Now we get to an example that has a bit too much noise Now we get to an example that has a bit too much noise. Note the multicolored peaks at 271, 273 and 279, the oddly-spaced interstitial peaks near 291 and 301, and it is impossible to determine the real nucleotide is at 310. if your signal is extremely low (anything below G=50, but on some sequencers even G=100) and your peaks are uninterpretable, then you should consider the lane to be simply blank - i.e. a failed sequencing reaction. Don't try to read baseline noise for usable data!

II. Yanlış adlandırılmış pikleri kontrol edin Baz eşleşmelerinde ciddi hatalar var mı? Heterozigot pikler (2li tepecikler) var mı? Sometimes the computer will mis-call a nucleotide when a human could do better. Most often, this occurs when the basecaller calls a specific nucleotide, when the peak really was ambiguous and should have been called as 'N'. Occasionally, the computer will call an 'N' when a human would be confident in making a more specific basecall. Such mis-calls can occur even in the most error-free regions of the gel. Quickly scan the gel for extremely small peaks, 'N' calls, and any mis-spaced peaks or nucleotides.

1- Eşit aralıklı olmayan pikler This is a great example of why a weak sample, with its consequent noisy chromatogram, is untrustworthy.

2-Dubleks pikler

Base and ambiguity codes used in DNA FASTA-files The table below gives you the encoding for the four bases (A, C, T, G) and for ambiguous positions in your DNA-sequence. This one-letter-code is usually used in FASTA-Files and other DNA file formats. The etymology should give you a mnemonic to memorize the codes. IUBMB standard codes.

III. Jelin sonlarına doğru rezolusyon kaybı Çok iyi giden kromatogramlar bile sonlara doğru bozulmaya başlayabilir

The sequencer will continue attempting to "read" this data, but errors become more and more frequent Late in the chromatogram, watch for multiple bases of any one nucleotide where there really should be only one. Watch, too, for wide peaks mis-counted by the program as two nucleotides, when it should have been just one. Wide peaks may also obscure smaller adjacent peaks (no example shown here).

IV. Problemeler artmaya başladığında sekans sonlandırılmalı Yayınlanabilir sonuçlar için SNP arayışında Sekans kalitesini göz önünde bulundurarak sekans uzunluğu istenilenden daha kısa olarak alınabilir (1100 yerine 900 nükleotid gibi) An investigator trying to locate intron-exon boundaries won't mind fairly high error rates, but one who needs publishable sequence can accept only the best sequence. One who is doing a BLAST search to identify a coding sequence can accept fairly high error rates, and still obtain a match, but if you want to spot SNPs, you can use only the highest-quality sequence.

Diğer ortak problemler Spikes in the chromatogram (air bubbles on a dim chromatogram) A few peaks with multiple colors (PCR on polymorphic genomic region) The first few nucleotides are poor (typical - don't expect good sequence close to the primer) One base is [wrong/missing/inserted]. Did the sequencer screw up? (probably not, but sequence the other strand) There's an 'N' in the midst of otherwise good sequence

Problems with the Primers & PCR Cond. PCR is exponential, sequencing is not. PCR reactions can be tailored to the primer; sequencing reactions cannot. PCR reactions actually can use a mismatched priming site; sequencing rarely can. Sequencing PCR products with the PCR primers: With two primers replicating opposite strands, a PCR reaction is exponential. That has some HUGE effects on the results. Primers that are inefficient can still work for PCR. Most people amplify beyond the linear part of the reaction, meaning inefficient primers will produce as much product as efficient ones. For sequencing, this is NOT the case. Inefficient primers will give only weak bands. Also - and this is often very important - PCR will amplify even if the target DNA is only a small proportion of the DNA present. 99% of your plasmid prep may be some junk DNA, but the remaining 1% will amplify just fine. It will not sequence, however.

1. Nesil sekanslamanın limitasyonları Çok fazla klonlama adımı gerektiriyor Düşük işlem kapasiteli İlk insan genomunun sekanslanması; 3 milyar $ ~10 yıl >20.000 BAC clones

Next generation sequencing (Yeni nesil sekanslama) Schadt et al., 2010; 2. nesil (yıka-görüntüle metodolojisi) 2005 Birbiri ile aynı olan sekansların hibritleşmesi ve uygun yıkama ve görüntüleme protokolleri (template immobilization)

Kalıp DNA immobilizasyon stratejileri Metzker et al., 2010; Illumına solexa

Kalıp DNA immobilizasyon stratejileri Metzker et al., 2010; HELICOS MOVIE

Kalıp DNA immobilizasyon stratejileri Metzker et al., 2010; PASICIF BS MOVIE

2. Nesil sekans platformları Roche Pyrosequencing(2004) Illumina/Solexa(2006) Applied Biosystems Solid sequencer(2007) Helicos(2009)

Roche 454 pirosekanslama (pyrosequencing) İlk ticari yeni nesil sekans platformu (2004) Prosedür adımları; DNA kütüphanesi hazırlanması emülsiyon PCR (isolation of single DNA molecules in water-oil droplets) sekanslama

Roche 454 pyrosequencing Mardis et al., 2008;

Roche 454 pyrosequencing Mardis et al., 2008;

Roche 454 pyrosequencing Mardis et al., 2008;

Roche 454 pyrosequencing emPCR produces ~1m copies of Metzker et al., 2010; emPCR produces ~1m copies of DNA template at the surface of each bead Step 1: A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin. Step 2 The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide. Step 3 ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated. Step 4: Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added. Step 5 Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATP S) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the Pyrogram® trace. Each dNTP incorporation leads to Ppi release and ultimately light production via luciferase

MOVIE Pyrosequencing ROCHE 454

Illumina/Solexa Mardis et al., 2008;

Illumina/Solexa Mardis et al., 2008; Solid-phase amplification

Illumina/Solexa Mardis et al., 2008;

Illumina/Solexa Metzker et al., 2010; CCD(charge-coupled device) camera takes images after each nucleotide incorporation

MOVIE Illumina/Solexa http://www.youtube.com/watch?v=77r5p8IBwJk http://www.youtube.com/watch?v=HtuUFUnYB9 Y&feature=related https://www.youtube.com/watch?v=fCd6B5HRa Z8

Yeni nesil sekans platformlarının karşılaştırması Metzker et al., 2010;

2. Nesil sekanslamanın limitasyonları Pek çok yıkama ve görüntüleme basamağı Ortalama okuma uzunluğu 35-400 baz arasında Karmaşık örnek hazırlama PZR çoğaltmalına geriksinim duyulması sekanslama hatalarını ortaya çıkartıyor Uzun süre

Güncel teknolojiler 2. nesilden 3. nesile geçiş; Helicos BioSciences Munroe et al., 2010; 2. nesilden 3. nesile geçiş; Helicos BioSciences Ion torrent-semiconductor technology

Helicos BioSciences Metzker et al., 2010; Single molecule templates involved, no PCR Immobilized primer, Poly(A) adaptor CCD(charge-coupled device) camera takes images after each nucleotide incorporation

MOVIE HELICOS http://www.youtube.com/watch?v=TboL7wODBj 4

Ion torrent-semiconductor technology Munroe et al., 2010; Bazlar bağlandıkça açığa salınan H+ iyonunun algılanması metodu Sonuca ulaşma süresi kısa ucuz Hala yıka-görüntüle teknolojisi

MOVE ION TORENT http://www.youtube.com/watch?v=yVf2295JqU g

1., 2. ve 3. nesil sekanslamaların karşılaştırılması

NGS Genomics and Applications Ancient genomes sequenced (mammoth, neanderthal) Protein-DNA interactions via CHIP-seq Noncoding RNA discovery Mutation discovery from a programmable microarray and sequencing them using NGS Characterization of biodiversity with the help of sequenced genomes Variability search in human genome (CNV, SNP, epigenetics)

References Schadt E. E., Turner S., Kasarskis A. 2010 A Window into Third Generation Sequencing Hum Mol Genet. Krivanek, O.L., Chisholm, M.F., Nicolosi, V., Pennycook, T.J., Corbin, G.J., Dellby, N., Murfitt, M.F., Own, C.S., Szilagyi, Z.S., Oxley, M.P. et al. Atom- by-atom structural and chemical analysis by annular dark-field electron microscopy. Nature, 464, 571-574. http://www.pacificbiosciences.com/index.php?q=evolution-of-sequencing Mardis E.R. 2008 Next-Generation DNA Sequencing Methods Annu. Rev. Genomics Hum. Genet. 9:387–402 Metzker M.L. 2010 Sequencing technologies- the next generation Nature Reviews 11:31-46 Munroe D.J., Harris T.J.R. 2010 Third-generation sequencing fireworks at Marco Island. Nature Biotechnology 28:426-428 Genome sequencing: the third generation 2009 Nature News 457:768-769 Eid J., Fehr A., Gray J. et al. 2009 Real-Time DNA sequencing from single polymerase molecules. Science 323:133-138 Bowers J., Mitchell J., Beer E. Et al. 2009 Virtual Terminator nucleotides for next generation DNA Sequencing Nature Methods 6(8): 593–595 Branton D., Deamer D.W., Marziali A. 2008 The potential and challenges of nanopore sequencing Nat Biotechnol. 26(10): 1146–1153