a, DNA fragments are generated, for example, by random shearing and joined to a pair of oligonucleotides in a forked adaptor configuration. The ligated products are amplified using two oligonucleotide primers, resulting in double-stranded blunt-ended material with a different adaptor sequence on either end. b, Formation of clonal single-molecule array. DNA fragments prepared as in a are denatured and single strands are annealed to complementary oligonucleotides on the flow-cell surface (hatched). A new strand (dotted) is copied from the original strand in an extension reaction that is primed from the 3' end of the surface-bound oligonucleotide; the original strand is then removed by denaturation. The adaptor sequence at the 3' end of each copied strand is annealed to a new surface-bound complementary oligonucleotide, forming a bridge and generating a new site for synthesis of a second strand (dotted). Multiple cycles of annealing, extension and denaturation in isothermal conditions result in growth of clusters, each ~1um in physical diameter. This follows the basic method outlined in ref. 33. c, The DNA in each cluster is linearized by cleavage within one adaptor sequence (gap marked by an asterisk) and denatured, generating single-stranded template for sequencing by synthesis to obtain a sequence read (read 1; the sequencing product is dotted). To perform paired-read sequencing, the products of read 1 are removed by denaturation, the template is used to generate a bridge, the second strand is re-synthesized (shown dotted), and the opposite strand is then cleaved (gap marked by an asterisk) to provide the template for the second read (read 2). d, Long-range paired-end sample preparation. To sequence the ends of a long (for example, >1 kb) DNA fragment, the ends of each fragment are tagged by incorporation of biotinylated (B) nucleotide and then circularized, forming a junction between the two ends. Circularized DNA is randomly fragmented and the biotinylated junction fragments are recovered and used as starting material in the standard sample preparation procedure illustrated in a. The orientation of the sequence reads relative to the DNA fragment is shown (magenta arrows). When aligned to the reference sequence, these reads are oriented with their 5' ends towards each other (in contrast to the short insert paired reads produced as shown in a–c). See Supplementary Fig. 17a for examples of both. Turquoise and blue lines represent oligonucleotides and red lines represent genomic DNA. All surface-bound oligonucleotides are attached to the flow cell by their 5' ends. Dotted lines indicate newly synthesized strands during cluster formation or sequencing. (See Supplementary Methods for details.)
specialize in amino acid analysis and oligo synthesis
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