Loss of DNA due to size-selection
In this experiment, sample losses throughout the library construction process were minimized by 1) carefully optimizing dispensing and aspiration steps, 2) employing a “with-bead” strategy that obviates the transfer of DNA between plates for consecutive enzymatic steps, and 3) shearing input DNA to a size (~200 bp) that overlaps well with the range of adapter ligated fragment sizes (200–500 bp) captured during size selection. Unlike common ChIP-Seq protocols that recommend 18 cycles of amplification, we varied the number of cycles based on DNA input and calculated the number of amplification cycles required to produce 100 ng of library for sequencing on the Illumina Genome AnalyzerIIx. As shown in Table 3, 18 cycles of enrichment are expected to produce sufficient library material when 1 ng input ChIP DNA is used for library construction. For the 10 ng of input recommended in the Illumina protocol [6,], only 9 cycles should be needed to produce the desired quantity of material for sequencing.Discussion
NGS library preparation has become a standard procedure for many sequencing labs. Preparing high-quality libraries from limited amounts of input DNA remains a challenge, particularly when automated liquid handling is required to achieve high throughput. Whilst adapter-ligated, size-selected DNA can easily be amplified to levels required for sequencing, it is important to limit the number of PCR amplification cycles in any library construction workflow as far as possible to ensure the lowest possible incidence of duplicate reads, amplification bias (leading to uneven coverage), chimeric library inserts, nucleotide substitutions, and heteroduplex formation (due to thermocycling after substrate depletion), which can lead to inaccurate library quantification.
This Application Note demonstrates the successful automation of low-input library construction, using the KAPA High-Throughput Library Preparation Kit on the Eppendorf epMotion 5075 TMX. Our data have shown that it is possible to obtain sufficient amounts of size-selected library material for Illumina sequencing from as little as 1 ng of input DNA after 18 cycles of amplification with the engineered KAPA HiFi DNA Polymerase, or only 9 cycles beginning with 10 ng of input DNA. This presents a significant improvement over competitor solutions for the preparation of ChIP-Seq libraries.
In summary, the following factors are critical for successful, automated library construction from low-input DNA:
A reliable, optimized liquid handling workstation that requires minimal human intervention.
High-quality Eppendorf consumables (tubes, tips and PCR plates) that offer ultra-low affinity to nucleic acid, which is vital for samples that can be obtained only in trace amounts.
High-quality library construction reagents for efficient enzymatic reactions, combined with the optimized cleanup and dual-SPRI size selection parameters provided by the KAPA HTP Library Preparation Kit.
The “with-bead” strategy, which allows for recycling of SPRI beads in consecutive reaction cleanups, thereby minimizing DNA losses and reducing per sample cost.
The combined advantages of the Eppendorf epMotion 5075 TMX and the KAPA High-Throughput Library Preparation Kit offer an industry-leading solution for standard and low-input library preparation, particularly for ChIP-Seq. The automated workflow on the epMotion is easily scalable, to provide for the processing of up to 96 samples per run. Moreover, the spectrum of applications can be expanded to any other NGS library construction workflows that share the same basic steps. Data obtained from the 50 and 200 ng cDNA samples are good examples of utility in DNA and RNA sequencing applications.