DNA Sudoku—harnessing high-throughput sequencing for multiplexed specimen analysis

Abstract

Next-generation sequencers have sufficient power to analyze simultaneously DNAs from many different specimens, a practice known as multiplexing. Such schemes rely on the ability to associate each sequence read with the specimen from which it was derived. The current practice of appending molecular barcodes prior to pooling is practical for parallel analysis of up to many dozen samples. Here, we report a strategy that permits simultaneous analysis of tens of thousands of specimens. Our approach relies on the use of combinatorial pooling strategies in which pools rather than individual specimens are assigned barcodes. Thus, the identity of each specimen is encoded within the pooling pattern rather than by its association with a particular sequence tag. Decoding the pattern allows the sequence of an original specimen to be inferred with high confidence. We verified the ability of our encoding and decoding strategies to accurately report the sequence of individual samples within a large number of mixed specimens in two ways. First, we simulated data both from a clone library and from a human population in which a sequence variant associated with cystic fibrosis was present. Second, we actually pooled, sequenced, and decoded identities within two sets of 40,000 bacterial clones comprising approximately 20,000 different artificial microRNAs targeting Arabidopsis or human genes. We achieved greater than 97% accuracy in these trials. The strategies reported here can be applied to a wide variety of biological problems, including the determination of genotypic variation within large populations of individuals.[Supplemental material is available online at www.genome.org.]There are many biological applications wherein the sequence of a small region must be determined from many independent specimens and wherein the link between the specimen and the sequence must be preserved. Examples might include studies of variation within specific regions from a large population, looking for correlations between variants and phenotype in areas that might have been narrowed by prior linkage analysis, or even medical diagnostics. We were faced with the practical matter of determining clone identity and quality in a very large collection of arrayed clones derived from a pooled oligonucleotide mixture. Use of next-generation sequencing technologies (NGST) for interrogating sequence for any of these tasks is hampered if libraries must be constructed independently from each sample or if different barcodes must be applied to each specimen prior to sequencing, particularly if specimen numbers run into many thousands.To circumvent such limitations, we have developed a novel multiplexing framework in which the identity of individual specimens is not encoded directly by an appended barcode sequence. Instead, specimen identities are encoded by positional information created by an optimized series of pooling schema. In this manner, more than 100,000 different samples can be analyzed using only a few hundred barcodes (e.g., 384). Results Conceptual fr...

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