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Lotus DNA Library Prep Kit

One kit. Flexible workflow. Endless applications.

The Lotus DNA Library Prep Kit enables streamlined preparation of high-quality next generation sequencing (NGS) libraries from double-stranded DNA (dsDNA). The kit uses enzymatic fragmentation to generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing applications on Illumina platforms.

  • Get the uniform sample coverage you need without relying on expensive equipment
  • Regain valuable time with a fast, simple workflow
  • Create application-specific NGS libraries by adding IDT adapters and xGen products for target capture

The Lotus kit can be customized for your applications when combined with one of the many IDT adapter options (see Ordering section). Additionally, use of xGen hybridization capture products provides a complete NGS solution that takes you from sample preparation to sequencing.

Ordering

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Adapters

To complete your libraries, you will need adapters. xGen Stubby Adapter and Unique Dual Index Primer Pairs are pre-packaged in single-use formats to start experiments quickly. xGen Dual Index UMI Adapters–Tech Access are made to order for PCR-free sequencing. Alternatively, use our step-by-step Custom Adapter Configurator tool to create Custom NGS Adapters and solutions that fit your exact application.

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.

Figure 1. Overview of Lotus DNA library preparation. Our easy enzymatic method takes you from sample to sequencing while eliminating the need for acoustic shearing methods that require instrumentation and extra time.

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.

FeatureDetails
Kit sizes16 reactions
96 reactions

Sample types
(not provided)

Double-stranded DNA from fresh frozen tissue
Genomic DNA
Full-length double-stranded cDNA
DNA input range1–250 ng
Suggested DNA insert sizeWhole-genome applications:  350 bp
Hybridization capture:  200 bp
TA-ligation adapters
(see Table 2)
Full-length:  PCR amplification is optional
Stubby (truncated):  PCR is required to add index sequences

Adapters

Complete your library prep with our selection of ready-to-use xGen Dual Index UMI Adapters or xGen Stubby Adapter and UDI Primer Pairs (Table 2). We also offer many high-quality, custom adapters and index primers that are application-specific and ideal for use with the Lotus kit.

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

ApplicationBest choiceRationale
Whole genome sequencing, metagenomics, PCR-free sequencing (≥100 ng input)xGen Dual Index UMI Adapters–Tech AccessFull-length adapters – required for PCR-free applications
Whole-genome sequencing, metagenomics with PCR (1–250 ng input), exome sequencing, and targeted germline sequencing (SNVs, indels)xGen Stubby Adapter and UDI Primer PairsSimple workflow – prepare your ligation master mix with a universal adapter and introduce unique dual index sample barcodes via PCR              
Low-level mutation detection, down to ~1% frequencyxGen Dual Index UMI Adapters–Tech AccessMore sensitive – use UMI consensus analysis to remove sequencing errors
RNA-seq starting with full-length, double-stranded cDNA inputxGen Dual Index UMI Adapters–Tech AccessMore quantitative – UMI is used to remove PCR duplicates

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
Figure 2. Overview of hybridization capture sequencing workflow.

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).

Figure 3. Uniform genome coverage for PCR-free and PCR-amplified libraries. Libraries were prepared using 100 ng of human gDNA (NA12878, Coriell) using full-length UDI-UMI adapters (IDT) or amplified using 4 cycles of PCR and sequenced on the MiSeq® (PCR-free) and HiSeq® 4000 (amplified) platforms (Illumina). Normalized coverage of each library is shown as dark and light blue lines, the expected normalized coverage of 1.0 is indicated with a dotted line, and the number of 100 bp regions at each GC% is shown as a histogram in grey.

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).

Figure 4. Consistent coverage across a range of inputs using a custom xGen Lockdown panel for somatic variant detection. DNA libraries (10, 25, and 250 ng) were created from human genomic DNA (NA12878, Coriell) using full-length adapters and enriched for a 75 kb target region using a custom xGen Lockdown Panel. The enriched libraries were sequenced on a NextSeq® instrument (Illumina) and subsampled to 500,000 reads. Mean target coverage was consistent across all inputs (n = 6 for each condition).
Figure 5. 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 stubby adapters 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.

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.

Figure 6. 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.

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