(custom, coding exome and whole genome)
Sanger sequencing performed on an ABI3730xls has been optimized using a Beckman FX robot to ensure pipetting accuracy and use the minimum volume of reagents.
Consequently, pricing is kept low ~$1/sequence (250-500 nucleotides). General applications include variant detection (mutations or SNPs), exon and gene sequencing to validate next-generation sequencing results.
Next generation sequencing platforms include SOLiD 5500xls and Ion Proton semiconductor sequencing. The SOLiD5000xls enables 100 base paired-end sequencing of DNA fragment libraries. Agilent custom-capture designed target arrays are used to interrogate regions of interest…whether exons, whole genes or genomic loci. Typically 0.5-2.0Mb DNA custom capture panels are designed to generate data for ~2000 samples at high read depth >100x. Samples are barcoded and run in sets of 96, the reads bioinformatically de-convoluted prior to genome alignment. Subsequent annotation of variant call files includes data on read depth, putative pathogenicity and allele frequencies with comparison to public and propriety databases. We have a series of custom panels developed including a 0.5Mb set of 172 genes genetically linked to neurodegeneration that is especially informative for atypical diagnoses of parkinsonism and dementia. Running costs for 0.5Mb panels are ~$120/sample.
Alternatively, we use Ion Proton semiconductor sequencing for 58Mb whole human exome sequencing. Longer reads are an advantage over most technologies, typically 150-230 bases, which leads to a dramatic improvement in read alignment, depth and consequently annotation accuracy. AmpliSeq capture requires less starting material, typically ~100ng and reagent costs for 58Mb exome sequencing are ~$500/sample.