While whole genome sequencing stands as the most comprehensive method for genomic studies, labor-intensive bottlenecks during library preparation demand more flexible and streamlined workflows.
This whitepaper shows how an automated liquid handler can simplify library preparation for whole genome sequencing workflows.
Download this application note to discover:
- A library preparation workflow to generate high quality, sequencing ready libraries
- An easy-to-setup automated platform requiring minimal user training
- How to reduce the likelihood of human error and maximize walk-away time
Easy Illumina® Nextera® DNA FLEX Library Preparation using the epMotion® 5075t Automated Liquid Handler WHITE PAPER No. 13WHITE PAPER I No. 13 I Page 2 Solutions & Benefits Workflow: Library preparation in just 4 steps On the epMotion, Nextera DNA Flex users have full flexibility over which processes they wish to automate and from which steps they may want to proceed. The entire workflow is divided into only 4 methods for processing up DNA Extraction from Saliva DNA Extraction from Blood to 96 samples in parallel as shown in Figure 1. The epMotion can accommodate different sample inputs and provides full f lexibility as to which kit version (i.e. 24-plex or 96-plex) the user chooses. Genomic DNA Extraction Method 1s 50 µl Tip consumption: 16-16 300 µl Tip consumption: 104-384 Run Time: 35-87 minutes Tagmentation DNA purification Method 2 DNA Extraction from Bacterial Colonies 50 µl Tip consumption: 56-216 300 µl Tip consumption: 128-416 Run Time: 74-108 minutes Indexing Amplification DNA purification Pooling Method 3 50 µl Tip consumption: 88-304 300 µl Tip consumption: 120-480 Run Time: 54-98 minutes Method 4 50 µl Tip consumption: 32-104 300 µl Tip consumption: 0 Run Time: 6-9 minutes Figure 1: Workflow for the Nextera DNA Flex Library Preparation protocol on the epMotion showing single steps. Red boxes show which steps are part of which method file. Steps highlighted in green are not part of the qualified method but can be performed using the epMotion. The running times on the epMotion and tip consumption are shown for handling 24 and 96 samples for each method. The complete process allows construction of 96 sequencing-ready libraries in less than a day with hands-on time of less than one hour.WHITE PAPER I No. 13 I Page 3 Figure 2: Libraries generated by the epMotion are highly comparable. The figure shows the 2100 BioAnalyzer® High Sensitivity DNA chip results for 8 samples using the Nextera DNA Flex Library Prep kit automated on the epMotion with the gel-view (left) and electropherogram overlay (right). Gel Electropherogram Size [bp] Ladder Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Generate high-quality libraries with the automated workflow on the epMotion When investing into a method, it is important to have as little variation as possible and to reduce the risk of wasting costly reagents and labor time. The epMotion allows the generation of ready to sequence libraries with minimal setup time. To demonstrate the reliability of the method 8 libraries were prepared from 200 to 300 ng DNA from Coriell Institute female reference cell line NA12878 and sequenced in two independent experiments starting at Method 2. The average fragment size was closely clustering around ~600 bp as recommended by Illumina (Figure 2). Library yields were highly reproducible and comparable to manual data (Table 1). Furthermore, the median sequencing insert size clustered tightly around the 350 bp optimum and is comparable to manual library preparations (Table 1). This translated to good sequencing performance data with high read diversity and even coverage across the genome and autosomes and a low coefficient of variance (CV). Table 1: Comparison of results obtained with manual and automated library preparation. Data presented is an average of two 8-plex runs on a HiSeqX® system, with sequencing reads trimmed to a 30X depth (380 million reads, 2 x 151 bp). Data analysis performed using the BaseSpace Sequence Hub Whole Genome Sequencing v5.0 App. Manual Preparation Automated epMotion® 5075t Yield (Qubit), ng/µL 13.2 13.6 Yield CV (%) 5.1 3.5 Index CV (%) 10.8 11 Median insert size range (bp) 352 339 Mean diversity > 2.3e9 >3.0e9 Autosome mean coverage (Fold) 30 - 32x 30 - 32x Autosome Callability (%) 95.0 95.5