Abstract
BackgroundTo complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance.ResultsHere we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA.ConclusionsAluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.
Highlights
To complement next-generation sequencing technologies, there is a pressing need for efficient presequencing capture methods with reduced costs and DNA requirement
Individual Alu-transposons in the human genome are on the average only 15 - 20% divergent from each other, and PCR primers complementary to the Alu consensus sequence have been employed for inter-Alu PCR [14,15,16,17,18]
Various primer combinations containing both H-type and T-type primers, allowing the amplification of intervening sequences between two Alu heads, between two Alu-tails as well as between one head and one tail, readily yielded a smeared gel indicating the presence of a wide diversity of amplicons of different sizes
Summary
To complement next-generation sequencing technologies, there is a pressing need for efficient presequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. Alu elements were once considered as ‘junk DNA’, their biological importance, in particular their influence on genome instability is being increasingly recognized [2,3]. They are abundant in gene-rich regions [4,5], exert a major impact on genomic architecture [6], and increase local recombination rates [7]. Neighborhoods of Alu-transposons are a highly significant sequence subset of the human genome in terms of evolutionary development and pathogenesis
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