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 microarrays1 and oligonucleotide libraries2.
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.
LC Sciences has been synthesizing oligonucleotide libraries for use in capture applications since 20043. Our OligoMix® product is a pooled library of thousands of oligonucleotides with customer specified sequences. The product was developed in response to the need for pooled oligonucleotides used in multiplexing reactions and we now have several customers that are using OligoMix® in sequence capture applications that they have developed.
Recently, researchers from the National Human Genome Research Institute conducted a systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing4. They report the testing, optimizing, and rigorous comparing of three genomic enrichment methods: Solution Hybrid Selection (Gnirke et al. 2009) Microarray-based Genomic Selection1, and Molecular Inversion Probes (MIP)2. Oligonucleotides for MIP were obtained from both Agilent and LC Sciences and tested as separate pools. HapMap DNA samples were used for testing and they found that that all three genomic enrichment methods are highly accurate (>99.998%) and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.
- Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME. (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4(11), 907-09.
- 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.
- 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.
- Teer JK, Bonnycastle LL, Chines PS, Hansen NF, Aoyama N, Swift AJ, Abaan HO, Albert TJ; NISC Comparative Sequencing Program, Margulies EH, Green ED, Collins FS, Mullikin JC, Biesecker LG. (2010) Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res 20(10), 1420-31.