Automated target enrichment using SeqCapEZ DNA kits on ACSIA NGS Capture Edition

Transcription

1 Application Note Automated target enrichment using SeqCapEZ DNA kits on ACSIA NGS Capture Edition Anh Thu VU, PrimaDiag Parc Biocitech, 102 Avenue Gaston Roussel, Romainville, France Magdalena NAWARA, Roche Diagnostics France 2 Avenue du Vercors, Meylan, France magdalena.nawara@roche.com

2 Abstract Being aware of the bottleneck of libraries preparation and target enrichment steps of next generation sequencing protocols, PrimaDiag developed the ACSIA NGS Capture Edition automated pipetting platform. With the ACSIA platform, you can efficiently automate these most laborious and time consuming manipulations with a very cost-effective methods using the lowest quantity of reagents and tips boxes. The validated method uses target enrichment (SeqCap EZ) provided by Roche Diagnostics. In this document, the technique used on the ACSIA NGS Capture workstation is described and some comparative efficiency results between automated and manual experiments are given. Introduction & Hardware description High-throughput sequencing, also known as Next Generation Sequencing (NGS), well described by its name, permits sequence a very large number of genes of many individuals per run. It is now a major driver in genetics research, providing a powerful tool to study DNA and RNA samples. Over the last few years, NGS technology has steadily progressed with increased throughput and decreased costs. For the purpose of saving analysis time and finding a more cost-effective approach by selectively sequencing chosen regions of a human genome, Roche Diagnostics developed the SeqCapEZ system which enables enrichment of a whole exome or a customized region. Nevertheless the target enrichment approach requires a very time consuming and laborious library preparation (minimum of 4 working days for a well-trained technician to prepare 48 libraries 8 multiplex). Because of a critical role of the library quality and several important considerations, the whole process (library preparation by using KAPA kit included) requires the technician to be highly concentrated during the manipulations (library preparation, target capture, heat of the reagents and samples, incubation for a specific time ). Given these constraints, it is evident that automated target capture is requested by most laboratories. To meet this demand, PrimaDiag developed the NGS Capture system based on ACSIA platform using the latest technologies which allow the handling of magnetic beads, temperature control with 0.1 C of accuracy and shaking from 200 to 3000 rpm. The ACSIA platform and the automation protocols provided by PrimaDiag allow the enrichment of up to 48 multiplex by using SeqCap EZ kit. All liquid handling parameters are carefully optimized for minimal reagents consumption and the protocols are optimized to use a minimum number of tips (3 tip boxes for preparing 48 multiplex instead of 9). Keywords: ACSIA, PrimaDiag, Roche, automation, liquid handling, automated pipetting platform, NGS, library preparation, target enrichment, Capture, Exome, SeqCap, KAPA. Materials : ACSIA NGS Capture Edition Bioanalyzer (Agilent) CLARIOstar (BMG Labtech) Human DNA (Provided by Metabolic Unit of the Genetic Center at the Pitié-Salpétrière hospital) SeqCap EZ Library kit (Roche Diagnostics) Figure 1: ACSIA NGS Capture Edition 2

3 The strategy for SeqCap capture depends on the size of target region. For capturing targets smaller than 100kb, the double capture is favored for high performance. For a larger target or exome enrichment, the single capture is preferred. In the experiment presented below, we studied a panel of 18 genes representing 67kb, the strategy for capture chosen is double capture. In this document, we describe the methods used and demonstrate the efficiency of the double capture with SeqCap EZ library SR kit on the ACSIA NGS Capture Edition system when compared to the manual method. Methods Figure 2: Worktable of ACSIA NGS in capture by SeqCap EZ method 1. Washing and Recovering Captured multiplex DNA sample Before starting on ACSIA NGS Capture system, the method for PrimaController II software named SeqCapEZlibrarySR.prws is loaded to the control computer. The labwares and reagents have to be placed onto the ACSIA worktable as shown in Figure 2. Click to validate button for choosing the number of multiplex need to be processed in this run (from 1 to 48 pools): If the number of multiplex DNA sample is not changed compared to the launched protocol, choose Continue & Ignore, If the number of multiplex DNA sample is not the same, choose Continue & Update. 3

4 The system gives the quantity of each reagents necessary in each rack/reservoir for performing this protocol with the chosen number of multiplex. Firstly, the machine prepares wash buffer need to be equilibrated at 47 C by diluting. During the heat of these buffers, ACSIA system prepares the Captures beads by washings 3 times with dedicated buffer. Once capture beads are ready, samples are brought to the capture beads. 45 minutes of incubation at 47 C is performed on the machine. A homogenization by vortexing at 1000 rpm is done at 15-minute intervals to ensure that the beads remain in suspension. During 45 minutes of incubation, the machine prepares other wash buffer for saving time. After the incubation, the multiplex samples are washed 3 times with wash buffer at 47 C and 3 times at room temperature. Finally, DNA target is eluted in 50µL of PCR-grade water. A LM-PCR with presence of capture beads is performed in the thermocycler. The pools are returned back to ACSIA for a final clean up by magnetic beads. For simple capture experience, at the end of the previous purification, the libraries could be quantified and checked for size distribution before loading to the sequencer. In this experiment, the double capture is performed. After the previous purification, the same hybridization then washing and recovering captured multiplex DNA sample as described above are implemented following by a LM-PCR and purification by magnetic beads. DNA target is eluted from beads in 50µL of PCR-grade water. 2. Quality control of libraries To check the quality of the library preparation, a quantification by ClarioSTAR system then a quality check by Bioanalyzer for ensuring the size distribution of fragments before sequencing on Illumina MiSeq platform. In this experiment, the double capture was studied, for this method, a qpcr for enrichment measurement is not necessary. However, it s strongly recommended for simple capture by SeqCapEZ of Roche NimbleGen. Results & discussions 1. Quality control post-capture After 2 hybridizations following by 2 captures, the average fragment size falls between bp for 2 experiments (manual and automated) as required by Roche NimbleGen. 4

5 However, a tighter profile is observed with the pool prepared by ACSIA robot (Figure 3) which demonstrated a better purification of samples during the process of library preparation by using KAPA DNA kit. Figure 3: Quantification post-capture by Bioanalyzer of multiplex DNA sample prepared respectively on ACSIA (above) and manually (below) 2. Sequence results: Method Sample Total reads ACSIA Manual Correctly mapped reads Inside the capture Outside the capture Duplication level ,91% 90,68% 8,23% 17,03% ,80% 87,47% 11,34% 23,62% ,05% 91,15% 7,90% 18,28% ,22% 89,92% 9,31% 24,68% ,34% 87,74% 11,60% 20,20% ,18% 91,10% 8,08% 17,42% ,92% 91,01% 7,92% 18,10% ,08% 91,42% 7,66% 17,04% ,65% 91,36% 7,29% 17,03% ,93% 90,92% 8,02% 19,72% ,05% 91,60% 7,44% 17,61% ,29% 89,71% 9,58% 23,28% ,78% 90,74% 8,04% 30,23% ,05% 91,01% 8,04% 16,95% ,12% 91,13% 7,99% 17,21% ,25% 89,22% 10,02% 29,43% ,05% 86,78% 12,27% 23,24% ,01% 90,24% 8,77% 17,11% ,31% 91,19% 8,12% 32,47% ,06% 90,68% 8,38% 30,07% ,28% 90,67% 8,61% 26,12% ,08% 89,65% 9,43% 25,83% ,15% 87,55% 11,59% 20,51% ,93% 91,33% 7,60% 17,34% Figure 4: Mapping statistics of samples prepared respectively by ACSIA and manually 5

6 Samples prepared by ACSIA and manually are sequenced on MiSeq platform. The reads were then demultiplexed and mapped. The table in the figure 4 shows the mapping statistics of tested samples. The number of bases correctly mapped of each sample demonstrates a quality similar between automated and manual preparations. The number of total reads are quite similar. The percentage of aligned bases falls under 98.65% and 99.34% for automated preparation and 98.78% and 99.31% for manual preparation. Regarding the duplication level in the same figure, this value is globally a little bit lower and has a better homogeneity for sample prepared by ACSIA (From to 24.68%) than manually (From to 32.47%).In the figure 5 below, we show the quality of covered regions, one more time, we noticed the similar result obtained from the automated and manual preparations. The number of bases that contain at least one low-covered position (<50X) is 0. Almost every bases are a very good coverage depth, only around 14 to 17 bases of automated preparation and 9 to 25 bases of manual preparation have a coverage lower than 500X. Method Sample Number of regions Number of base < 500 < 200 < 100 < 50 < 40 < 30 < 20 < 10 < ACSIA Figure 5: Depth 0 of coverage 0 of interested 0 regions Manual

7 Figure 6: Depth of coverage of multiplex DNA prepared on ACSIA The figure 6 above shows the median and minimal coverage of 12 samples prepared on ACSIA platform. Figure 7: Depth of coverage of multiplex DNA prepared manually The figure 7 shows the coverage depth obtained of 12 samples prepared manually. 7

8 Figure 8: Depth of coverage of 18 captured genes of samples prepared on ACSIA (blue) and manually (red) The design of probes for this experiment permits to capture a panel of 18 different genes, the figure 8 shows their coverage depth in the sequence of samples prepared by ACSIA (in blue) and manually (in red). We observed that the depth is quite close between automated and manual preparation. However, the automated preparation gives a coverage depth tightly deeper and more homogeneous. We remind also that in these experiment, all expected mutations were detected in 12 samples automated and manually prepared. Definitions/Abreviations : MWU: Magnetic Work Unit DNA: Deoxyribonucleic Acid PCR: Polymerase Chain Reaction SPRI: Solid Phase Reversible Immobilization CONCLUSION Thanks to ACSIA NGS Capture platform, the most laborious steps which are the capture by Streptavidine beads then clean-up can be efficiently automated. These automations permit you to save up to 4 hours. The number of tips used for each sample is divided by 3 (3 tip boxes are used for automated protocol compared to 9 tip boxes for manual protocol). The obtained results from automated and manual preparation are quite similar. Nevertheless, the automated result shows a slightly better profile postcapture and sequencing. Acknowledgement : This study was completed thank to Dr. Christine BELLANNE CHANTELOT and her team at Genetic center. 8

9 REFERENCES 1] PrimaDiag ACSIA User Manual [2] KAPA Library Preparation Kit for Illumina platforms [3] High-Throughput NGS Library Preparation Technical Guide. KAPA [4] SeqCap EZ Library SR User s Guide [5] F. Mertes, A. ElSharawy. Targeted enrichment of genomic DNA regions for next-generation sequencing [6] S.Fisher, A. Barry. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries [7] Double Capture: An alternative protocol for sequence capture of difficult targets. Roche [8] Agencourt AMPureXP, Protocol v001. PCR Purification. 9