Editing NGS Library Preparation (section)

== The genome Analyzer II workflow ==

consists of sample preparation, cluster amplification,<br/>sequencing and data analysis.<br/>Sample preparation

enome Analyzer II can utilize nucleic acids from various resources if it is provided in a<br/>compatible format (de Magalhães, Finch & Janssens 2010, Schmidt et al. 2009,<br/>Linnarsson 2010). The library consists of short fragments of double-stranded DNA.<br/>These fragments should be approximately 150 to 500 base pairs long to have uniform<br/>performance in cluster amplification. The fragments have adapters on both ends of the<br/>fragment complementary to both adapters on flow cell and sequencing primers<br/>(Linnarsson 2010, Pettersson, Lundeberg & Ahmadian 2009, Mardis 2008).<br/>The primary step in standard library preparation protocol is fragmentation in which the<br/>genomic DNA is sheared to short fragments of double-stranded DNA. The fragments that<br/>have single-stranded overhangs at this point are then end repaired to generate blunt-<br/>ended fragments. The addition of deoxyadenosine monophosphate (dAMP) to the 3’-end

of the strands enhances the efficiency of the correct ligation (Linnarsson 2010). The<br/>polymerase chain reaction (PCR) amplification is also needed in order to enrich the<br/>fragments that have been successfully prepared and thus have the adapters in both ends<br/>and are capable of amplifying and attaching to a flow cell. (Figure 1)

<br/>Fragmentation<br/>The fragmentation can be performed either by mechanical or sonar force or by enzymatic<br/>reaction. The official Illumina sample preparation protocol uses pressurized air based<br/>method called nebulization to fragment the DNA. Other means for fragmentation are<br/>focused sonication (Covaris Inc., Woburn, MA, USA) and enzymatic fragmentation with<br/>Fragmentase (New England Biolabs, Ipswich, MA, USA). In enzymatic fragmentation<br/>random nicks are created by nuclease and the nicks are recognized by endonuclease<br/>which cleaves the other strand (New England Biolabs 2010). Also DNaseI in the<br/>presence of Mn 2+ fragments the DNA (Linnarsson 2010).<br/>Sample yields from focused sonication can be more than four-fold compared to<br/>nebulization since the fragment size distribution is narrower. The enzymatic methods<br/>have wider size distribution than sonication but have better yields than nebulization<br/>(Linnarsson 2010).

<br/>End repair<br/>After the fragmentation the fragments have 3’ and 5’ overhangs which must be cut off in<br/>order to have blunt-ended fragments which can be further prepared for adapter ligation.<br/>T4 DNA polymerase catalyzes the synthesis of DNA in the 5’ -> 3’ direction thus filling<br/>the 5’ overhangs and cleaves the 3’ overhangs with its 3’ -> 5’ exonuclease activity<br/>(Orkin 1990).

A-tailing<br/>The aim of incorporating deoxyadenosine monophosphate (dAMP) to the 3’ end of the<br/>blunt-ended DNA fragment is to prevent the formation of concatemers in ligation and to<br/>enhance the ligation efficiency. The adapters have complementary deoxythymidine<br/>monophosphate (dTMP) in the 3’ end of the ligating end of the adapter which enables<br/>successful ligation of the adapter. The deoxyadenosine triphosphates (dATP) are<br/>incorporated by Klenow fragment which is an N-terminal truncation of DNA Polymerase<br/>I which retains polymerase activity, but has lost the 5 ́→ 3 ́ exonuclease activity (Clark,<br/>Joyce & Beardsley 1987).



Ligation<br/>The ligation process incorporates adapters to both ends of the blunt-ended DNA<br/>fragments. Successful ligation should result in different adapters in different ends of the<br/>fragments. A dTMP on the 3’ end of the ligating end of the adapter enables the ligation to<br/>the DNA fragment with 3’ end dAMP overhang. The DNA strands of the adapters are<br/>complementary only on the ligating end of the adapter resulting in Y-shape. This<br/>significantly reduces the formation of fragments with the same adapter in both ends of<br/>the fragments (Linnarsson 2010). The phosphorotioate modifications in the ends of the<br/>adapters protect the adapters from endonucleases. The ligation is performed by T4 DNA<br/>ligase. T4 DNA ligase is an enzyme purified from E. coli C600 pcl857 pPLc28 lig8. It is<br/>capable of catalyzing the phosphodiester bond formation of juxtaposed 5' phosphate and<br/>3' hydroxyl termini of DNA and RNA. It can incorporate both blunt-ended and cohesive-<br/>ended termini. (Engler, Richardson 1982)



Purification<br/>After each sample preparation step the library must be purified from salts, enzymes,<br/>surplus of adapters and other reagents. There are several approaches for purification from

DNA in a high-salt buffer binds to silica-gel- membrane. The additional substances are<br/>removed, and the DNA eluted in a low-salt buffer. This method is susceptible to pH<br/>changes. (Hamaguchi, Geiduschek 1962, Vogelstein, Gillespie 1979)<br/>Gel filtration with resin<br/>Gel filtration purification utilizes different method compared to the other purification<br/>approaches. As in other methods DNA is bound to beads or membranes, in gel filtration<br/>additional substances in the solution are separated from DNA during gel filtration.<br/>Primers, short single stranded DNA strands, salts, buffers, deoxyribonucleotide<br/>triphosphates (dNTP) and other small molecules are eluted in larger volume than large<br/>molecules such as DNA. (EdgeBio 2010)<br/>Paramagnetic bead purification<br/>Solid Phase Reversible Immobilization (SPRI) beads have polystyrene core coated with a<br/>layer of iron (Figure 2). The iron grants the beads their paramagnetic properties. The<br/>surface of the bead is a polymer layer containing carboxyl functional groups. The SPRI<br/>beads bind the negatively charged nucleic acid with the carboxyl groups leaving other<br/>substances in the solution. The beads carrying the nucleic acid can be separated from the<br/>solution by external magnetic field. This enables efficient washing and elution of the<br/>DNA or RNA in proper elution buffer. (Beckman Coulter Genomics 2010, Deangelis,<br/>Wang & Hawkins 1995)