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[h=2]Introduction[/thanks]Over recent years, the cost of sequence acquisition has reduced dramatically, owing to significant advances in next generation sequencing (NGS) technologies. As a result, the use of sequence acquisition is becoming increasingly common in new settings such as the clinic, small research groups, and contract research organizations. The widespread adoption of NGS seems likely despite the fact that most liquid handling workstations have been validated to complete the library preparation protocols. As NGS requires a high number of sample manipulations, liquid handling automationis needed to ensurereliable and consistent results.
[h=2]VERSA Mini NGLP Workstation[/thanks]
[h=2]Methods and Materials[/thanks]The template used for library preparation was gDNA isolated from the Pocket Mouse (Chaetodipus intermedius). A Covaris instrument was used for fragmentation, which was divided into two equal parts. One part was processed in an automated fashion using the VERSA Mini NGLP (automated library), while the other was processed manually using the Illumina TruSeq library preparation kit (manual library). The NEXTflex DNA Sequencing Kit from Bioo Scientific was used for enzymatic modifications and for completion of the purification steps.
The VERSA workstation handled the reaction setup for dA tailing, end repair, barcoded adapter ligation as well as incubations and magnetic bead cleanups. Purified, sequenceable libraries (one manual and one automated) were size selected through agarose gel electrophoresis and excision of a 400 to 500 bp target range. Finally, amplification was performed.
The competency of both the manual and automated libraries was assessed. The distribution of fragment size was analyzed using a Bioanalyzer trace from Agilent Technologies. DNA concentrations were ascertained using the PicoGreen reagent from Life Technologies. A qPCR using a KARA Biosystems Library Quant Kit measured the levels of fragments that were bound to the adapter. Following cluster generation and sequencing of the libraries using a single lane on a HiSeq 2000 run, bioinformatic analysis was performed to characterize the data.
[h=2]Results and Discussion[/thanks]
A qPCR experiment using Library Quant Kits was performed to ensure that the library fragments were properly ligated to Illumina adapter sequences. The C1 values as well as the concentration of adapter-bound fragments for manual and automated libraries were very similar and of acceptable quality for sequencing.
All of the control filtration measures put in place by the University of Arizona were passed by both libraries. In order to validate the libraries as representative of the original template and of the integrity required to provide raw, usable data, they were submitted to DNA cluster generation and sequencing on one lane of a HiSeq 2000 instrument.
In order to prove that the automated technique was feasible, the sequence characteristics needed to be examined. A FastQC analysis of the R1 read from the paired-end sequences showed that sequence content and quality were similar for the two libraries. The overall read quality scores (Figure 4) and per base sequence quality showed that both sample preparations resulted in high integrity libraries. Furthermore, both data sets reflected an overall GC content of 39%. R2 sequence metrics reflected those found in R1.
Both manual and automated libraries seemed to have levels of sequence duplication that may have been introduced during the PCR amplification process. In addition, FastQC analysis indicated that in contrast to the number of unduplicated reads, the automated and manual data sets had minimum duplication levels of 77.3% and 92.7%, respectively, for the R1 reads (Figure 5). There was no significant difference between these levels for the R2 reads.
[h=2]Acknowledgement[/thanks]Produced from articles authored by Matthew J. Nesbitt, Matthew Kaplan, Sikander Gill, and Dong Liang.
[h=2]About Aurora Biomed[/thanks] is a worldwide leader in the design and development of lab automation solutions for life science, environmental science, drug discovery/safety and chemical analysis research. We are committed to improving the quality of human and environmental health by providing products and services which facilitate a higher sample throughput while improving quality, accuracy and precision.
Aurora's product range includes automated liquid-handling equipment, atomic absorption spectrometers, atomic fluorescence spectrometers, ion channel screening technology and microwave digestion systems, increasing the efficiency of sample management in a wide range of research applications. We are headquartered in Vancouver, BC, Canada, and have global sales, support, and service offices.
[h=2]VERSA Mini NGLP Workstation[/thanks]
Figure 1. The VERSA Mini NGLP Workstation
To this end, the application of the VERSA Mini NGLP Workstation (Figure 1) for NGS library preparation has been validated by the University of Arizona Genetics Core. Since VERSA is an open-platform workstation, third party reagent kits can be used, which significantly reduces the per sample cost. Moreover, the VERSAware user software allows complete control over aspiration and dispensing speeds as well as tip positioning that can be customized to precisely handle the genomic sample.[h=2]Methods and Materials[/thanks]The template used for library preparation was gDNA isolated from the Pocket Mouse (Chaetodipus intermedius). A Covaris instrument was used for fragmentation, which was divided into two equal parts. One part was processed in an automated fashion using the VERSA Mini NGLP (automated library), while the other was processed manually using the Illumina TruSeq library preparation kit (manual library). The NEXTflex DNA Sequencing Kit from Bioo Scientific was used for enzymatic modifications and for completion of the purification steps.
The VERSA workstation handled the reaction setup for dA tailing, end repair, barcoded adapter ligation as well as incubations and magnetic bead cleanups. Purified, sequenceable libraries (one manual and one automated) were size selected through agarose gel electrophoresis and excision of a 400 to 500 bp target range. Finally, amplification was performed.
The competency of both the manual and automated libraries was assessed. The distribution of fragment size was analyzed using a Bioanalyzer trace from Agilent Technologies. DNA concentrations were ascertained using the PicoGreen reagent from Life Technologies. A qPCR using a KARA Biosystems Library Quant Kit measured the levels of fragments that were bound to the adapter. Following cluster generation and sequencing of the libraries using a single lane on a HiSeq 2000 run, bioinformatic analysis was performed to characterize the data.
[h=2]Results and Discussion[/thanks]
Figure 2. Bioanalyzer traces for the automated (a) and manual (b) libraries. A dark electropherogram band in (a) proves the automated process efficiently recovered DNA within the range targeted by the size selection.
Bioanalyzer traces for both automated and manual libraries suggested acceptable fragment size distribution profiles, as shown in Figure 2. Traces were similar between the libraries. The automated library had an average fragment size of 417 bp, compared with an average size of 423 bp for the manual library. A PicoGreen experiment showed that the total DNA concentrations for the manual and automated libraries were 180.66 nM and 157.16 nM, respectively (Table 1).Table 1. Determination of total dsDNA concentration (PicoGreen) and adapter-bound fragments (qPCR).
| Library | PicoGreen (nM) | qPCR (nM) |
| Automated | 157.16 | 169.2 |
| Manual | 180.66 | 172.5 |
All of the control filtration measures put in place by the University of Arizona were passed by both libraries. In order to validate the libraries as representative of the original template and of the integrity required to provide raw, usable data, they were submitted to DNA cluster generation and sequencing on one lane of a HiSeq 2000 instrument.
Figure 3. Log (ARn) vs. cycle count for qPCR analysis of adapter-bound fragments in the automated (a) and manual (b) libraries. A common threshold of 32.7 was used for both, with Ct values of 5.3 and 5.0 in (a) and (b), respectively.
[h=2]Raw Sequence Data[/thanks]The raw sequence data obtained for 100 bp paired-end reads were organized by barcode, changed to FastQ files, and processed using a trimmer called Trimmomatic. Then, adapter sequences were removed and leading and trailing bases were scanned. A sliding window was employed to trim reads at points over which the median Q scores fell to below 15.In order to prove that the automated technique was feasible, the sequence characteristics needed to be examined. A FastQC analysis of the R1 read from the paired-end sequences showed that sequence content and quality were similar for the two libraries. The overall read quality scores (Figure 4) and per base sequence quality showed that both sample preparations resulted in high integrity libraries. Furthermore, both data sets reflected an overall GC content of 39%. R2 sequence metrics reflected those found in R1.
Figure 4. Per base and per sequence Q scores for R1 ends of the automated (a, b respectively) and manual (c, d respectively) libraries. All median per base scores exceeded Q28, and the average read quality peaked at Q38 for both libraries.
Discrepancies between the data sets of the two libraries were observed. The automated set had 75% more paired-end reads than that of the manual set, as shown in Table 2. This difference could be due to an extremely efficient DNA cluster generation with the automated library.Table 2. Paired-end reads for automated and manual data sets.
| Library | Reads (M) |
| Automated | 114.1 |
| Manual | 65.4 |
Figure 5. Sequence duplication of R1 reads for the automated (a) and manual (b) data sets. The ratio of unique to duplicate reads is higher in the automated data set than the manual set.
[h=2]Conclusion[/thanks]The is suitable for preparing DNA libraries on the Illumina platform that are as competent as those produced using manual techniques. Automation also helps in scaling the levels of sample preparation, eliminates complicated work, and saves valuable time.[h=2]Acknowledgement[/thanks]Produced from articles authored by Matthew J. Nesbitt, Matthew Kaplan, Sikander Gill, and Dong Liang.
[h=2]About Aurora Biomed[/thanks] is a worldwide leader in the design and development of lab automation solutions for life science, environmental science, drug discovery/safety and chemical analysis research. We are committed to improving the quality of human and environmental health by providing products and services which facilitate a higher sample throughput while improving quality, accuracy and precision.
Aurora's product range includes automated liquid-handling equipment, atomic absorption spectrometers, atomic fluorescence spectrometers, ion channel screening technology and microwave digestion systems, increasing the efficiency of sample management in a wide range of research applications. We are headquartered in Vancouver, BC, Canada, and have global sales, support, and service offices.
