To 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. Here 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 transposon...[ Read more ]

To 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. Here 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. AluScan, 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 enable an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement. Moreover, it 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.

The hexamer insertion (I) – deletion (D) polymorphism marker, namely rs71305152 amplified by the AluScan, was genotyped in Chinese cohorts of glioma (n = 362), non-glioma cancer (n = 354) and non-cancer control (n = 463) by direct sequencing and gel electrophoresis of PCR products spanning the polymorphic site. The marker parent gene FOG2 expression in glioma tissues (n = 72) of different grades was quantified by real-time RT-PCR. The results showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.020). Frequencies of the two homozygous (II, DD) genotypes were both significantly higher in gliomas (P = 0.021). The heterozygous (ID) genotype compared of the combined homozygous was more frequent in controls (P = 0.007) or non-glioma cancers (P = 0.026) than in gliomas. FOG2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.001), and of astrocytoma subtype (P = 0.002), with lower expression in the high-grade gliomas. The expression of FOG2 mRNA was significantly higher (P = 0.011) for the subgroup of surviving patients displaying a longer survival period compared to deceased patients with a shorter survival period. These findings suggest that FOG2 is a glioma susceptibility gene, in which the hexamer insertion-deletion provides a convenient marker for diagnostic genotyping. Moreover, FOG2 expression may serve as a severity indicator useful for glioma prognosis and a target for glioma therapeutics.