High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. DNA sequencing technology has benefited from tremendous progress over the past several years, with many platforms routinely producing >109 nucleotides (nt) of data during a single run [1]. Current generation high-throughput sequencers require a library of amplicons from which reads are generated randomly by a number of different strategies, including pyrosequencing [2], reversible chain-terminator expansion [3], and ligation [4]. Several FNDC3A strategies create brief reads fairly, in the number of 36C70 nt [5], in comparison to 1000413-72-8 supplier traditional Sanger sequencing which generates reads >800 nt long [6] regularly, [7]. For a few applications, such as for example microRNA evaluation [8], ChIP-Seq [9], or SAGE (Serial Evaluation of Gene Manifestation) [10], brief reads are adequate. Nevertheless, for resequencing known genomes [5] and set up of unfamiliar sequences [11], [12], brief reads present a bioinformatics problem and make adequate target sequence insurance coverage difficult to accomplish. To day, experimental answers to these issues have centered on two techniques: increasing the amount of reads created from an example or increasing read length. Complex advances such as for example paired-end reads [13], [14] or marketing of sequencing systems with hardware, software program, and/or reagent upgrades may raise the true amount of reads created from a test. Alternatively, extra reads could be made by sequencing an example multiple times simply. However, reaching sufficient coverage of focus on sequences with these 1000413-72-8 supplier solutions can be expensive. Coverage with short-read systems may also be improved by straight increasing examine length, which is achieved by increasing the number of synthesis or ligation cycles performed during sequencing. While lengthening reads does not necessarily incur additional cost, in practice, the signal to noise ratio of current technologies decreases at each cycle much more rapidly than in traditional Sanger sequencing, effectively limiting the number of bases that can be read with an acceptable degree of accuracy [3], [15]. We describe and demonstrate here a simple method for improving high-throughput short-read sequencing results using a cost-effective sample preparation technique. This process, termed 1000413-72-8 supplier the long march, utilizes a Type IIS restriction enzyme that cleaves DNA distal to its recognition motif [16], [17]. By embedding this recognition motif in the sequencing primer adapter of the initial amplicon library, iterative rounds of digestion and ligation produce a nested set of sub-libraries for sequencing. While we demonstrate this method using the Illumina (Solexa) GA2 platform, the long march procedure is applicable to any short-read shotgun sequencing system, including the ABI SOLiD and Helicos. We show that the long march increases contig length and absolute coverage (compared to the same number of reads produced without the procedure) using a cDNA library generated from genome assembly applications, based on relative enzyme efficiencies as well as starting DNA pool complexity. These results suggest that considerable improvements in absolute base 1000413-72-8 supplier coverage may be achieved through relatively simple and cost-effective modifications of high-throughput sequencing sample preparation protocols. In essence, the long march technique combines the desirable aspects of both shotgun sequencing and directed primer walking to produce substantially greater coverage within the same number of reads and using the same read length. Materials and Methods Long marching and barcoding bead-bound cDNA For with the following modifications: (1) the HBV sample used the adapters Sol-L-CC-RR (short-SolL-GsuI-CCRR and Sol-Adapter-L-short-phos-CC annealed), Sol-L-GG-RR (short-SolL-GsuI-GGRR and Sol-Adapter-L-short-phos-GG annealed), and Sol-L-TT-RR (short-SolL-GsuI-TTRR and Sol-Adapter-L-short-phos-TT annealed) for march rounds 1 through 3, and (2) PCR amplification of all marched aliquots was carried out for 15 cycles instead of 10 cycles using the 1000413-72-8 supplier PCR conditions described for the initial HBV library in Components and Strategies S1. Solexa sequencing of lengthy and preliminary marched cDNA For genome launch 5.4 [23] or even to the HBV genome (accession quantity: “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_003977″,”term_id”:”941241313″,”term_text”:”NC_003977″NC_003977) [21]. Any reads that didn’t match the genomes in a distinctive position.