Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing
© The Author(s). 2017
Received: 2 July 2016
Accepted: 26 February 2017
Published: 23 March 2017
The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library.
The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification.
The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated.
Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.
KeywordsPacBio SMRTbell NGS library preparation Bacterial genomic DNA Automation NGS workstation TapeStation System Bioanalyzer
Increased throughput from the use of next generation sequencing methods has revealed new information about the function and structure of bacterial genomes. The use of short reads to produce draft genomes leads to problems with GC content bias and repeat regions that make it tedious to produce closed genome assemblies. This technical note discusses the PacBio RS II approach using a single molecule, real-time DNA sequencing approach to improve genome assembly through extra-long read lengths. By reducing the number of contigs, the accuracy of the de novo assembly of bacterial whole genomes is facilitated. The real-time technology of the PacBio RS II allows determination of not only the full, closed, gDNA sequence, but also epigenetic modifications and plasmid DNA sequence simultaneously.
The 100K Pathogen Genome Project  is using the PacBio 10 kb SMRTbell Template Preparation kit to produce 1,000 closed genomes. The scale of this project required automation of the construction of the sequencing (SMRTbell™) library. To prepare libraries for sequencing in this way, gDNA must be cut into fragments to a target size of 10 kb. Critical to generating long sub-reads, it is important to start with high quality gDNA input in order to shear the gDNA into the target fragment size to ensure the correct concentrations during library construction to react properly with the concentrations of reagents in each of the given steps. Gel electrophoresis is a low-resolution traditional method with sizing against a ladder and determining concentration on an agarose gel by comparing peak density to a standard, and since it cannot be automated, is not suitable for a project of this size. Another way to measure size and concentration is to use the Agilent 2100 Bioanalyzer with the DNA 12000 assay, but the instrument only runs 12 samples at a time and cannot be automated. We will discuss the automation of preparation of libraries with the SMRTbell Template Preparation kit as well as analysis of gDNA, fragmented DNA and the final libraries ready for sequencing with both the Agilent electrophoresis platform: Agilent 2100 Bioanalyzer System using the DNA 12000 assay and the Agilent TapeStation System using the genomic DNA ScreenTape and matching reagents.
Genomic DNA was sheared using the Covaris g-TUBE device (520079) according to the manufacturer specifications . After fragmentation, DNA was evaluated with the TapeStation System with the Genomic DNA assay and also with the Agilent 2100 Bioanalyzer System with the Agilent DNA 12000 assay (5067–1508) [8, 9]. Both of these methods have minimal sample consumption and return both sizing and quantitation. The sheared gDNA sample input was normalized for all samples between 1–5 μg into library construction for PacBio SMRTbell 10 kb Library Preparation.
Determination of the quality of the gDNA
Fragment gDNA using a Covaris g-TUBE device
QC the sizing and adjust the concentration
Repair DNA damage and repair ends of fragmented DNA
Purify the DNA
Blunt-end ligate using blunt adapters
Purify template for submission to a sequencer
With the automation, this workflow takes about 7 h for post-shearing clean-up and library construction. Once the PacBio 10 kb library is made, the final library was confirmed with the Agilent 2200 TapeStation with the Genomic DNA ScreenTape assay and the Agilent 2100 Bioanalyzer System with the Agilent DNA 12000 assay to determine the size of the library. Libraries are quantified using a Qubit 2.0 Fluorometer (Q32866) with a Qubit dsDNA HS Assay Kit (Q32854, both from Invitrogen, Carlsbad CA) to measure the library concentration before submission to the sequencing facility. The sequencing facility anneals sequencing primer and binds polymerase to the SMRTbell templates before loading the library onto the PacBio RS II.
The genomic DNA isolated from four model organisms with a range of GC content were made into libraries prepared on the Agilent NGS Workstation with PacBio SMRTbell Template Preparation kit for sequencing on the PacBio RSII. Finished sequences showed GC content very close to the known GC content, thus showing this process produced minimal bias (Table 1).
The PacBio SMRTbell 10 kb Library preparation kit can be used with automation such as the Agilent Bravo to prepare microbial libraries with minimal GC bias. QC of the starting DNA and the required fragment preparation with the Covaris g-TUBE can be done with the Agilent 2200 TapeStation and the gDNA ScreenTape assay directly from the 96 well plates used by the Bravo to prepare the libraries.
DNA integrity number
Next generation sequencing
Single molecule, real-time
We gratefully acknowledge the technical assistance provided by Kerry Le, Sum Leung, Christina Kong, Lucy Cai, Alvin Leonardo, Vivian Lee, Surene Foutouhi and Patrick Ancheta. We thank the 100K Pathogen Genome Sequencing Project for providing the cultures to conduct the study.
Funding provided to BCW (NIH - 1R01HD065122-01A1; NIH - U24-DK097154; AGILENT TECHNOLOGIES THOUGHT LEADER AWARD, FDA - 5U01FD003572-04).
Availability of data and materials
All data was analyzed during this study are included in this published article.
NK isolated DNA, conducted experiments, analyzed TapeStation data, and wrote the manuscript; WN and KT conducted experiments and analyzed TapeStation data; RA & AW isolated DNA; KSK, JK and LH provided technical assistance with library preparation; LK conceived of experiments, analyzed data, and wrote the manuscript; SL provided programming of the automation to run the protocols; BCW conceived of experiments, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
Agilent Technologies provided test instruments and initial funding to BCW. Pacific Biosciences provided PacBio SMRTBell 10 kb Library Preparation Kit and sequencing.
Consent for publication
Ethics approval and consent to participate
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- 100K Pathogen Genome Project. 2013 [cited 2016 June 30]; Available from: http://www.100kgenomes.org.
- Kong N, et al. Production and analysis of high molecular weight genomic DNA for NGS pipelines using Agilent DNA extraction kit (p/n 200600). 2013. doi:10.13140/RG.2.1.2961.4807.Google Scholar
- QIAamp DNA Mini Kit. 2016 [cited 2016 June 30]; Available from: https://www.qiagen.com/us/shop/sample-technologies/dna/dna-preparation/QIAamp-DNA-Mini-Kit#resources.
- Greenspoon SA, et al. QIAamp spin columns as a method of DNA isolation for forensic casework. J Forensic Sci. 1998;43(5):1024–30.View ArticlePubMedGoogle Scholar
- Agilent Genomic DNA ScreenTape System Quick Guide (p/n G2964-90040). 2016 [cited 2016 June 30]; Available from: http://www.agilent.com/cs/library/usermanuals/Public/ScreenTape_gDNA_QG.pdf.
- Agilent 2200 TapeStation User Manual (p/n G2964-90002). 2016 [cited 2016 June 30]; Available from: http://www.agilent.com/cs/library/usermanuals/Public/G2964-90002_TapeStationPalpatine_USR_EN.pdf.
- Covaris. Covaris USER MANUAL: g-TUBE 2012 [cited 2016 June 30]; Available from: http://covarisinc.com/wp-content/uploads/pn_010154.pdf.
- Agilent 2100 Bioanalyzer User Manual (p/n G2946-90004). 2016 [cited 2016 June 30]; Available from: https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack).pdf.
- Agilent Technologies, I. Agilent DNA 7500 and DNA 12000 Kit Quick Start Guide. 2013 [cited 2016 June 30]; Available from: http://www.agilent.com/cs/library/usermanuals/Public/G2938-90025_DNA7500-12000_QSG.pdf.