Benchmarks
Optimization and cost-saving in tagmentation-based mate- pair library preparation and
sequencing Kaori Tatsumi, Osamu Nishimura, Kazu Itomi, Chiharu Tanegashima, and Shigehiro Kuraku Phyloinformatics Unit, RIKEN Center for Life Science Technologies, Kobe, Japan
BioTechniques 58:253-257 (May 2015) doi 10.2144/000114288 Keywords: mate pair library; genome assembly; genome scaffolding; CEGMA; tagmentation
Supplementary material for this article is available at
www.BioTechniques.com/article/114288.
In de novo genome sequencing, mate-pair reads are crucial for scaffolding assembled contigs. However, preparation of mate- pair libraries is not a trivial task, even when using one of the latest approaches, the Nextera Mate Pair Sample Prep Kit from Illumina. To reduce cost and enhance library yield and fidelity when using this kit, we have modified the manufacturer’s protocol based on (i) variable tagmentation conditions, (ii) intensive DNA shearing to decrease library insert length, and (iii) sequencing on an Illumina HiSeq with >150 cycles. Finally, we provide additional suggestions for further improvement in the application of this kit.
While the genome sequences of many traditional model species have become available, de novo sequencing of unexplored species remains challenging (reviewed in Reference 1). Particularly crucial is the use of mate-pair reads for joining contigs, a process known as genome scaffolding. Traditional approaches for obtaining mate-pair reads often require large amounts of genomic DNA, especially for libraries with long mate distances, as well as lengthy laboratory procedures (2,3). The release of the Nextera Mate Pair Sample Prep Kit (illumina, San Diego, CA), based on tagmentation technology, has significantly reduced the difficulty, preparation time
METHOD SUMMARY
Here we describe several key modification to the Nextera Mate Pair Sample Prep Kit, including optimizing DNA tagmen- tation conditions and improved coordination between insert size and sequence read length, that result in higher mate-pair library yield and fidelity while reducing costs.
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and cost of preparing mate-pair libraries (4). However, there remain opportunities to improve the efficiency and reduce costs for this preparation technique. Here we present several key modifications of the manufacturers protocol that address these issues. The first key step in our optimization
is a modification of the tagmentation conditions. The standard protocol provides only one condition with a fixed volume of tagment enzyme (12 l), but we performed the reaction with reduced volumes of the enzyme (4 and 8 l). We also tested self-made tagment buffer prepared according to another published protocol (5). For this test case, we used 4
g of genomic DNA from the Madagascar ground gecko Paroedura picta, quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA), in each reaction. The tagmented DNA was analyzed with pulse-field electrophoresis (Figure 1). The self-made buffer exhibited comparable tagmentation efficiency to the buffer supplied in the kit, while reactions with reduced volumes of enzyme yielded tagmented DNA with altered length distributions (Figure 1). The use of a reduced volume of enzyme and self-made buffer allows for a higher number of tagmentation reactions to be attempted using variable conditions. This strategy enables the identification of ideal reaction conditions that maximize the yield of DNA in the intended size range for any given genomic DNA sample. In particular, this modification, which yields larger DNA molecules, is advantageous in preparing mate-pair libraries spanning large distances (>10 kb), which tends to be hindered by low yields. In addition to the tagmentation step,
the strand displacement step employs some reagents whose amounts limit the number of preparations. To address this limitation, the strand displacement reaction was performed after size selection on a BluePippin (Sage Science, Beverly, MA) (see the Figure 2 legend)—not before size selection as in the standard protocol—which yielded a sufficient quantity of mate-pair libraries. Reversing the order of strand displacement and size selection enables significantly smaller volumes of strand displacement polymerase and buffer for reduced amounts of size-selected DNA, which allows for a larger number of preparations (up to 3-fold) than the standard protocol. This approach provides substantial cost savings, as other reagents for downstream steps are bundled for as many as 48 reactions, but the Nextera Mate Pair Sample Prep Kit is only intended for 12 reactions based on the “gel-plus” protocol. When preparing mate-pair libraries,
it has traditionally been a major concern that a high proportion of library inserts
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