Article

Decoding a Substantial Set of Samples in Parallel by Massive Sequencing

Science for Life Laboratory, Department of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), Solna, Sweden.
PLoS ONE (Impact Factor: 3.23). 03/2011; 6(3):e17785. DOI: 10.1371/journal.pone.0017785
Source: PubMed

ABSTRACT

There has been a dramatic increase of throughput of sequenced bases in the last years but sequencing a multitude of samples in parallel has not yet developed equally. Here we present a novel strategy where the combination of two tags is used to link sequencing reads back to their origins from a pool of samples. By incorporating the tags in two steps sample-handling complexity is lowered by nearly 100 times compared to conventional indexing protocols. In addition, the method described here enables accurate identification and typing of thousands of samples in parallel. In this study the system was designed to test 4992 samples using only 122 tags. To prove the concept of the two-tagging method, the highly polymorphic 2(nd) exon of DLA-DRB1 in dogs and wolves was sequenced using the 454 GS FLX Titanium Chemistry. By requiring a minimum sequence depth of 20 reads per sample, 94% of the successfully amplified samples were genotyped. In addition, the method allowed digital detection of chimeric fragments. These results demonstrate that it is possible to sequence thousands of samples in parallel without complex pooling patterns or primer combinations. Furthermore, the method is highly scalable as only a limited number of additional tags leads to substantial increase of the sample size.

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    • "Our approach to library preparation synthesized several published, successful , cost-saving strategies from the literature. We used a combinatorial sample tagging protocol (Neiman et al. 2011), which permitted the same set of barcoded primer pairs to be used for each indexed library whereby different combinations of barcodes and indices formed unique tags for each sample (Fig. 1). The protocol substantially reduced the complexity of sample handling as well as overall costs by eliminating the need for a unique PCR primer pair (~$60 per pair purified by high performance liquid chromatography) or a unique Illumina adapter (~$30 per adapter) per sample. "
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    • "Each target was amplified by a two-step PCR, and received a distinct index included in one of the inner primers (Figure 1b, Supplementary Table S3 and S4). The indices have been described previously2526, as well as the primers for TP53 exon 2 and 927 and the general primers for TP53 and Lambda20. Inner cycling, 94°C 5 min, [94°C 30s, 60°C 120 s, 72°C 3 min] × 10 cycles, 72°C 5 min, 4°C hold, outer cycling the same but 30 cycles. "
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    • "The incorporation of sample-specific barcodes, indexes or multiplex identifying sequences (MIDs) into sequencing libraries via ligation or PCR amplification is required for the identification of the original source of sequences. Double-barcoding strategies using a combination of two barcodes unique for each sample have been devised to reduce the probability of false assignment of sequencing reads to their sample origin (Galan et al., 2010; Neiman et al., 2011). For example, a double-indexing method, which incorporates barcodes into both ends of sample molecules via PCR amplification with 5 -tailed primers prior to library pooling, has been developed for improving the accuracy of multiplex sequencing on the current Illumina sequencing platform (Kircher et al., 2012). "

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