OligoMix^® Oligonucleotide Mixture - Technical Bulletin

Sequence Capture for Targeted Sequencing

Recent major advances in DNA sequencing technologies have resulted in several new “next-generation” platforms capable of generating massive amounts of reads very quickly and relatively inexpensively.  These new technologies hold the promise of one day, routinely sequencing entire complex eukaryotic genomes.

Currently, these new technologies do not have quite the through-put required yet for routine whole genome sequencing.  At this point, they are more effectively utilized for systematic study of genetic variation in certain targeted regions (subsets) of complex genomes.  By targeting specific regions, one can take better advantage of next-gen sequencing capability; essentially, more coverage is achieved by focusing reads on your area of interest.

However, selection of specific targeted areas is difficult due to the enormous size and complexity of an entire genome.  Other challenges include the large percentage of repeats or non-coding sequences and the wide distribution of genetic coding elements within the genome.

Highly multiplexed PCR has been used to a degree as a method for selecting targeted genomic regions, but this method has inherent limitations.  When scaled to the level required to take advantage of the through-put of new sequencing technologies, multiplexed PCR becomes extremely complicated, time and labor-intensive, and thus expensive.  Consequently, the rate-limiting step for large scale genetic variation studies has become sample preparation methods.

More recently, there has been significant progress toward eliminating the time, cost, and performance limitations imposed by PCR.  Two separate methods of “sequence capture” have emerged as better alternatives to PCR for targeted enrichment of sequencing samples: custom sequence capture microarrays^1,2 and oligonucleotide libraries³.

LC Sciences’ µParaflo^® technology is particularly well suited to provide sequence capture solutions by either of these methods through massively parallel synthesis of high quality DNA on biochips.  Thousands of customer specified oligonucleotide sequences are in situ synthesized on a programmable high density microfluidics chip.  Standard DMT-phosphoramidite chemistry and advance microfluidics design ensure high quality synthesis.  Genomics samples can be applied directly to the biochip via microfluidics for on-chip sequence capture or alternatively, the oligonucleotides can be cleaved from the chip and attached to magnetic beads via a 5’ terminal modification and mixed with genomic samples to perform sequence capture in solution.

1. Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., and Church, G. (2004) Accurate multiplex gene synthesis from programmable DNA chips.Nature 432, 1050-1054. [abstract]
2. Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, Gao Y, Church GM, Shendure J. (2007) Multiplex amplification of large sets of human exons. Nat Methods. 4(11), 931-36.  [abstract]
3. Hodges E, Xuan Z, Balija V, Kramer M, Molla MN, Smith SW, Middle CM, Rodesch MJ, Albert TJ, Hannon GJ, McCombie WR. (2007) Genome-wide in situ exon capture for selective resequencing. Nat Genet. 39(12),1522-27.  [abstract]

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