Lotus DNA Library Prep Kit

Applications

The Lotus DNA Library Prep Kit produces high-quality next generation sequencing (NGS) libraries that are suitable for many applications including:

• Whole genome sequencing (WGS)
• PCR-free sequencing
• Detection of germline inherited single nucleotide variations (SNVs) and insertions/deletions (indels)
• Hybridization capture of target sequences (e.g., the exome or transcripts of interest)
• Low frequency somatic variation detection of SNVs and indels
• RNA-seq starting with full-length, double-stranded cDNA input
• Metagenomic sequencing

Method

This kit involves minimal enzymatic incubations and bead-based purification steps, thereby reducing sample handling and overall library preparation time to under 2 hours. The major steps are depicted in Figure 1 and are summarized as follows:

• Enzymatic preparation. Fragmentation, end-repair, and dA-tailing of dsDNA are all performed in a single reaction.
• Ligation of either full-length or stubby P5 and P7 adapters. When using full-length adapters, the final PCR step is optional and can be used to increase library yield. However, stubby adapters (sometimes called truncated adapters) require amplification with primers to incorporate sample indexing sequences and to add the P5 and P7 sequences.
• Optional PCR amplification. Choose to amplify your libraries based on adapter and DNA input used. A bead-based cleanup removes oligonucleotides and small fragments after PCR.

Kit overview

The Lotus kit comes with all the buffers, reagents, and enzymes needed for fragmentation, adapter ligation, and an optional PCR (to add index sequences, depending on adapter type, or to increase the amount of DNA for sequencing). You supply the DNA and add adapters that fit your goals. Table 1 provides additional specifications for using this kit.

Table 1. Specifications of the Lotus DNA Library Prep Kit.

Adapters

The Lotus kit does not include adapters, giving you the flexibility to design and use your own adapter-indexing strategies. We offer many high quality, custom designed adapters and index primers that are application specific and ideal for use with the Lotus kit (Table 2).

Table 2. How to choose IDT adapters for common applications when using the Lotus DNA Library Prep Kit.

Complete workflow for hybridization capture experiments

Lotus DNA Library Prep Kit paired with adapters from IDT create libraries that are compatible with xGen hybridization capture probes and reagents for when you only need to perform targeted sequencing (Figure 2). Here is a list of the key components you will need:

• xGen Hybridization and Wash Kit
• xGen Universal Blockers—TS Mix or 10 bp TS Mix
• xGen Lockdown Panels and Probe Pools
• xGen Gene Capture Pools
• xGen Library Amplification Primers

Uniform GC coverage from PCR-free and amplified libraries

The Lotus DNA Library Prep Kit produces consistent genome coverage with low bias across samples (Figure 3).

Lotus DNA Library Prep offers superior coverage and uniformity for targeted sequencing

IDT adapters and xGen Lockdown Probes and Panels are manufactured using stringent, proprietary methods that are critical for producing high-quality oligos for NGS applications. When these adapters and hybridization capture probes are used with the Lotus Kit for targeted sequencing, results show consistent, highly uniform, sequence coverage (Figures 4–5).

Superior representation of 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 (Figure 6A). Consistent genome coverage was observed across samples with varying levels of GC content (Figure 6B). The variability in size of the genomes, input amount, and GC content do not influence results.

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