A simple library prep workflow for many sequencing applications

The Lotus DNA Library Prep Kit enables streamlined preparation of high-quality next generation sequencing (NGS) libraries from input DNA. The kit includes enzymatic fragmentation to generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing applications on Illumina platforms. Here, we describe how you can:

• Regain valuable time with a fast, simple workflow
• Create libraries for nearly any sequencing application
• Get the uniform sample coverage you need without relying on expensive equipment

Flexible workflow

The Lotus DNA Library Prep Kit allows the enzymatic step to be performed in a single tube, thereby reducing sample handling and overall library preparation time to approximately 2 hours. With this modular kit, you can customize your library for your application. For example:

• Fragment size—Incubation times are used to control fragmentation size. Fine-tune fragment size to whole genome sequencing or targeted sequencing applications using recommendations provided in the certificate of analysis.
• Adapters and indexing strategy—Ligation is used to attach P5 and P7 adapters (not included in the kit) using standard TA-library construction. Customize with full-length or stubby adapters and use any sample indexing strategy you choose.
• Flexible workflow—PCR is optional, depending on your adapter or sample input requirements.

Library prep for almost any application

Before DNA can be sequenced, it must be converted into a library. Libraries are collections of DNA that have been fragmented to the appropriate size and have had adapter sequences attached. Library preparation can influence the results of your NGS experiments. The Lotus DNA Library Prep Kit produces high-quality NGS libraries that are suitable for a variety of applications that use DNA as input. Several factors influence the quality of sequencing results. The DNA input amount and GC content can vary between samples, so sequence coverage must be consistent regardless of sample variation. Lack of sequence uniformity can require additional or deeper sequencing to ensure adequate coverage of all targets, increasing sequencing costs. When paired with the xGen Exome Research Panel for targeted exome sequencing, the Lotus Kit facilitates highly uniform coverage, eliminating the need for additional sequencing (Figure 1). When tested with metagenomic samples, representation of an artificial microbial community was consistent across a range of GC contents and input amounts (Figure 2). Other downstream uses for this kit include whole genome sequencing, variant detection, and PCR-free sequencing.

Figure 1. Highly uniform sequence coverage with xGen Exome Research Panel leads to lower sequencing costs. (A) A Lotus DNA library was created from 100 ng of human genomic DNA (Coriell) using TruSeq™–Compatible Stubby Adapters and Indexing Primers and was enriched using the xGen Exome Research Panel. The enriched libraries were sequenced on an NextSeq^® instrument (Illumina) and subsampled to 50M reads. The data shows deep, uniform coverage with a flanked on-target rate of 90.2% and a duplication rate of 5.3% (calculated with Picard). (B) Exons 1 and 2 of RB1 show uniform, complete coverage on the Integrative Genomics Viewer. RB1 is a gene that is often poorly covered using competitor's capture technologies.

Figure 2. Variability in size of genomes, input amount, and GC content do not influence results in a metagenomics analysis. (A) Libraries were prepared using the MSA-1000™ microbiome standard (ATTC, an artificial microbial community of 10 strains), Lotus kit, full-length adapters, and PCR amplification with P5 and P7 primers. (B) Libraries were prepared with 5 ng of MSA-1000 gDNA. Normalized genome coverage for 3 representative strains with varying GC% content is shown as colored lines The number of 100 bp regions at each GC% is shown as histograms.

Superior coverage and uniformity

No matter what you decide to do with your NGS libraries, you can count on consistent, uniform results. One test performed with the kit used an artificial microbial community of 10 strains. The observed representation of each genome was consistent across a range of inputs (1, 10, and 25 ng) and correlated well with expected results. Consistent genome coverage was observed across samples with varying levels of GC content (Figure 1). The variability in size of the genomes, input amount, and GC content do not influence results. This kit is compatible with a range of DNA inputs
(1–250 ng) and exhibits low bias to achieve uniform sequence coverage. The Lotus DNA Library Prep Kit can be used in conjunction with IDT adapters, including Illumina TruSeq™-Compatible Custom Adapters (Stubby or Full-length), xGen Stubby Adapter and UDI Primers Pairs, and xGen UDI-UMI Adapters. IDT adapters and xGen Lockdown Probes and Panels are manufactured using stringent, proprietary methods that are critical for producing high-quality oligonucleotides for NGS applications. Whether you choose to vary the input, adapters, or end use, the Lotus DNA Library Prep Kit will give you high-quality results.

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