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The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing.

Sorber K, Chiu C, Webster D, Dimon M, Ruby JG, Hekele A, DeRisi JL - PLoS ONE (2008)

Bottom Line: High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information.Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension.We also offer a theoretical optimization of the long march for de novo sequence assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. Despite producing gigabases of sequence per run, these technologies still present obstacles in resequencing and de novo assembly applications due to biased or insufficient target sequence coverage. We present here a simple sample preparation method termed the "long march" that increases both contig lengths and target sequence coverage using high-throughput short-read technologies. By incorporating a Type IIS restriction enzyme recognition motif into the sequencing primer adapter, successive rounds of restriction enzyme cleavage and adapter ligation produce a set of nested sub-libraries from the initial amplicon library. Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension. We demonstrate the utility of the long march in resequencing of the Plasmodium falciparum transcriptome, where the number of genomic bases covered was increased by 39%, as well as in metagenomic analysis of a serum sample from a patient with hepatitis B virus (HBV)-related acute liver failure, where the number of HBV bases covered was increased by 42%. We also offer a theoretical optimization of the long march for de novo sequence assembly.

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Related in: MedlinePlus

Iterative rounds of GsuI digestion and barcoded adapter ligation create nested sub-libraries.Adapter flanked cDNA molecules are attached to streptavidin beads via biotin modification of the Sol-S adapter. Yellow triangles indicate the GsuI recognition motif engineered into the Sol-L adapter, while the connected black arrow represents the distal cut site. Adapter barcodes and corresponding reads are classified as AA (green), CC (red), or TT (blue). Reads from the initial library and all three long march steps are aligned to form an 84 bp contig.
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pone-0003495-g001: Iterative rounds of GsuI digestion and barcoded adapter ligation create nested sub-libraries.Adapter flanked cDNA molecules are attached to streptavidin beads via biotin modification of the Sol-S adapter. Yellow triangles indicate the GsuI recognition motif engineered into the Sol-L adapter, while the connected black arrow represents the distal cut site. Adapter barcodes and corresponding reads are classified as AA (green), CC (red), or TT (blue). Reads from the initial library and all three long march steps are aligned to form an 84 bp contig.

Mentions: To initiate the long march procedure, RNA from Plasmodium falciparum was reverse transcribed into double-stranded cDNA, biotinylated, and bound to streptavidin beads (see Materials and Methods S1). In construction of the initial library, the adapter containing the sequencing primer hybridization site (Sol-L) was modified before its NN overhang to incorporate the recognition motif of the Type IIS restriction enzyme GsuI (5′-CTGGAG-3′). Each march round began with digestion of the bead-bound cDNA with GsuI, which cleaves double-stranded DNA 14 nt distal to this motif (Figure 1) [16], [17]. Digested cDNA was then ligated to barcoded Sol-L adapters, and this digestion and ligation process was repeated iteratively to generate three nested sub-libraries in addition to the initial cDNA library. The initial library contained no barcode while subsequent rounds were barcoded AA, CC, and TT, respectively. After 5–10 cycles of PCR, the initial library and each sub-library was clustered and sequenced in a separate Illumina (Solexa) GA2 flow cell lane.


The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing.

Sorber K, Chiu C, Webster D, Dimon M, Ruby JG, Hekele A, DeRisi JL - PLoS ONE (2008)

Iterative rounds of GsuI digestion and barcoded adapter ligation create nested sub-libraries.Adapter flanked cDNA molecules are attached to streptavidin beads via biotin modification of the Sol-S adapter. Yellow triangles indicate the GsuI recognition motif engineered into the Sol-L adapter, while the connected black arrow represents the distal cut site. Adapter barcodes and corresponding reads are classified as AA (green), CC (red), or TT (blue). Reads from the initial library and all three long march steps are aligned to form an 84 bp contig.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2566813&req=5

pone-0003495-g001: Iterative rounds of GsuI digestion and barcoded adapter ligation create nested sub-libraries.Adapter flanked cDNA molecules are attached to streptavidin beads via biotin modification of the Sol-S adapter. Yellow triangles indicate the GsuI recognition motif engineered into the Sol-L adapter, while the connected black arrow represents the distal cut site. Adapter barcodes and corresponding reads are classified as AA (green), CC (red), or TT (blue). Reads from the initial library and all three long march steps are aligned to form an 84 bp contig.
Mentions: To initiate the long march procedure, RNA from Plasmodium falciparum was reverse transcribed into double-stranded cDNA, biotinylated, and bound to streptavidin beads (see Materials and Methods S1). In construction of the initial library, the adapter containing the sequencing primer hybridization site (Sol-L) was modified before its NN overhang to incorporate the recognition motif of the Type IIS restriction enzyme GsuI (5′-CTGGAG-3′). Each march round began with digestion of the bead-bound cDNA with GsuI, which cleaves double-stranded DNA 14 nt distal to this motif (Figure 1) [16], [17]. Digested cDNA was then ligated to barcoded Sol-L adapters, and this digestion and ligation process was repeated iteratively to generate three nested sub-libraries in addition to the initial cDNA library. The initial library contained no barcode while subsequent rounds were barcoded AA, CC, and TT, respectively. After 5–10 cycles of PCR, the initial library and each sub-library was clustered and sequenced in a separate Illumina (Solexa) GA2 flow cell lane.

Bottom Line: High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information.Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension.We also offer a theoretical optimization of the long march for de novo sequence assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. Despite producing gigabases of sequence per run, these technologies still present obstacles in resequencing and de novo assembly applications due to biased or insufficient target sequence coverage. We present here a simple sample preparation method termed the "long march" that increases both contig lengths and target sequence coverage using high-throughput short-read technologies. By incorporating a Type IIS restriction enzyme recognition motif into the sequencing primer adapter, successive rounds of restriction enzyme cleavage and adapter ligation produce a set of nested sub-libraries from the initial amplicon library. Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension. We demonstrate the utility of the long march in resequencing of the Plasmodium falciparum transcriptome, where the number of genomic bases covered was increased by 39%, as well as in metagenomic analysis of a serum sample from a patient with hepatitis B virus (HBV)-related acute liver failure, where the number of HBV bases covered was increased by 42%. We also offer a theoretical optimization of the long march for de novo sequence assembly.

Show MeSH
Related in: MedlinePlus