High Quality DNA Isolation Suitable for Ultra Rapid Sequencing
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1) Modifications were made to decrease the time for whole genome sequencing (WGS) including faster DNA isolation, optimized sample preparation, sequencing and bioinformatics analysis. 2) DNA isolation using the chemagic MSM I instrument was optimized to isolate DNA from 24 blood samples in 1.5 hours, yielding 40 μg/mL of high quality DNA suitable for sequencing. 3) Sequencing on an Illumina HiSeq 2500 was modified for ultra rapid runs, taking approximately 20 hours to sequence a genome to 40x coverage while maintaining over 90% of bases above Q30. Alignment and variant detection using Isaac software took less than 3.5 hours.
![Ultra Rapid Protocol
Fig. 5: Modified ultra rapid WGS.
Modifications were made to all aspects of WGS. The largest
decrease in time was accomplished by implementing Isaac for
alignment. Run times were decreased ~5 hours by modifying
the chemistries and cycle times of a standard HiSeq2500 in
rapid mode. Further modest time savings in sample preparation
were accomplished by using the KapaHyper library prep kit
omitting the final PCR amplification step. In total, including
DNA isolation WGS may be completed in <35 hours, including
analysis.
Sensitivity / Specificity
Sensitivity and Specificity are calculated using NA12878,
which has been highly characterized for SNPs and indels.
With ~40x coverage from two flowcells in ultra rapid mode
configuration, sensitivity and specificity were both >99%.
Tab. 3: Calculation of sensitivity and specificity.
Conclusion
The chemagic MSM I (PerkinElmer) enables fast DNA isolation
of high volume blood samples by coincidentally providing
high quality DNA suitable for sequencing. Modifications to
all aspects of Whole Genome Sequencing have led to a 40%
decrease in the time.
• Importantly, ultra rapid run mode does not affect run
quality scores or variant detection sensitivity and specificity.
• Further modifications will enable WGS to be completed
in <24 hours [1].
Acknowledgements
This work was supported by the Marion Merrell Dow
Foundation, Children’s Mercy - Kansas City,
W.T. Kemper Foundation, Pat & Gil Clements Foundation,
Black & Veatch, and NICHD and NHGRI (U19HD077693).
References
1. Miller NA, Farrow EG, Gibson M, Willig LK, Twist G,
Yoo B, Marrs T, Corder S, Krivohlavek L, Walter A,
Petrikin JE, Saunders CJ, Thiffault I, Soden SE, Smith LD,
Dinwiddie DL, Herd S, Cakici JA, Catreux S, Ruehle M,
Kingsmore SF. 2015. A 26-hour system of highly
sensitive whole genome sequencing for emergency
management of genetic diseases. Genome Med.
Sep 30;7(1):100.
Author Affiliations
1
Center for Pediatric Genomic Medicine,
2
Department of Pediatrics, and
3
Department of Pathology, Children‘s Mercy - Kansas City,
Kansas City, Missouri 64108, USA
4
School of Medicine, University of Missouri-Kansas City,
Kansas City, Missouri 64108, USA
• Consent Blood Sample
0
• DNA Isolation
1.5
• Sample QC/Dilution/Shearing
1
• Library Preparation
1.5
• Sample QC
1.5
• Sequencing Run
20
• Isaac Alignment
1
• Variant Calling
0.3
• RUNES
0.5
• Preliminary Results
PerkinElmer chemagen Technologie GmbH
Arnold-Sommerfeld-Ring 2
52499 Baesweiler, Germany
Phone: +49 (0)2401 805 501
Fax: +49 (0)2401 805 519
info@chemagen.com, www.chemagen.com
2702149NA12878 -
633 + 634
7747 3216 479489 3181638 99.71% 99.33%
True + False - False + True - Total Sens. Spec.
PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
Phone: (+1) 800-762-4000
or (+1) 203-925-4602
www.perkinelmer.com
CT72141601-01 Printed in Germany
Copyright ©2016, PerkinElmer Inc. All rights reserved. PerkinElmer®
is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.](https://image.slidesharecdn.com/apphqdnaisolationforngsrowlowresct72141601-170117142736/85/High-Quality-DNA-Isolation-Suitable-for-Ultra-Rapid-Sequencing-4-320.jpg)
High Quality DNA Isolation Suitable for Ultra Rapid Sequencing
- 1. For Research Use Only. Not for use in Diagnostic Procedures. Nucleic Acid Isolation Introduction Whole Genome Sequencing (WGS) necessitates high quality nucleic acids as well as rapid sequencing and pipelining for most effective implementation. In a pilot study we have previously demonstrated the utility of 50 hour rapid WGS. Herein, we present how fast DNA isolation and modifications to an Illumina® HiSeq® 2500 instrument and optimization of basecalling and variant detection using Isaac software v1 decrease sample preparation and sequencing to less than 35 hours. In order to achieve a minimum run time of 20 hours for a 40x genome (~120GB), cluster generation is completed in 1.5 hours, with an average cycle time of 6.5 minutes. Runs were 2x100 paired end, using v1 chemistry. Importantly the decrease in time did not affect quality scores, with ≥ 90% of data above Q30. Alignment and variant detection are done from BCL files using Isaac software v1 on a single compute node. Variant annotation is completed using custom software, RUNES (Rapid Understanding of Nucleotide variant Effect Software). The total bioinformatics pipeline takes less than 3 hours for completion. Taken together, the modifications made to sequencing and bioinformatics analysis have resulted in an approximately 40% decrease in the amount of time to complete a WGS. High Quality DNA Isolation Suitable for Ultra Rapid Sequencing Authors: Neil Miller1 , Emily Farrow1,2,4 , Margaret Gibson1 , Laurel Willig1,2,4 , Sarah Soden1,2,4 , Carol Saunders1,3,4 , Shane Corder1 , Greyson Twist1 , Stephen Kingsmore1,2,4 Children‘s Mercy - Kansas City, Kansas City, Missouri 64108, USA School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri 64108, USA James Richardson, Kevin Hall Illumina Inc., Chesterford Research Park, Little Chesterford, Essex CB10 1XL, UK Antonia Konczwald PerkinElmer chemagen Technologie GmbH, 52499 Baesweiler, Germany A P P L I C A T I O N N O T E
- 2. Methodology In order to further the decrease time to complete Whole Genome Sequencing, modifications were made to: • DNA Isolation • Sample Preparation • Sequencing • Alignment and Variant Detection DNA Isolation DNA isolation was performed using the chemagicTM MSM I instrument (PerkinElmer). The instrument is designed to isolate nucleic acids from diverse sample materials in three different configurations: (LV) low volume configuration, (MV) medium volume configuration, (HV) high volume configuration. Associated with the configuration is the sample number that can be maximally extracted per sample set and a sample volume range as indicated in Tab. 1 below. Tab. 1: chemagic MSM I instrument sample volume configurations. To meet further automation and quality control needs, the systems can be extended with chemagic QA Software and the chemagic Dispenser unit. These come along with pre-installed protocols and allow LIMS-compatible bar code reading/sample tracking as well as automated buffer filling for all volume applications. The chemagic MSM I instrument uses patented magnetic bead based technology for DNA isolation. Rotating needles (rods) and a switchable magnet facilitate an efficient sample mixing and resuspension of DNA bound to magnetic particles. This technology is very beneficial for high sample volumes from 0.2 - 10 mL, resulting in high yields and purities of the isolated DNA within a fast process. Fig. 2: The chemagicTM Separation Technology is based on the use of metal rods that are lowered into a process solution (A). To collect beads from the solution, the rods are magnetized. Pellets form at the tips of the rods, and the rods are withdrawn from the solution with the pelleted beads attached. Resuspension into the next process solution, for example, wash or elution buffer is achieved by switching off the magnetism while rotating the rods (B). This normally difficult step is thus performed quickly and thoroughly, resulting in isolation products with high yields and purities. Fig. 3: PerkinElmer chemagic MSM I instrument, equipped for the isolation of 24 medium vol. blood samples/run at Children’s Mercy - Kansas City. The current configuration of the instrument at Children’s Mercy allows for the fast and reliable isolation of 24 medium volume blood samples (200 µL - 4 mL whole blood) in only 1.5 hours. The average yield of DNA is 40 µg/mL. MAGNET OFF ROTATION ON B A MAGNET ON ROTATION OFF • Consent Blood Sample 0 • Sample Preparation 4.5 • Sequence Genome 25.5 • Analysis and Interpretation 16 • Preliminary Results Fig. 1: 50 hour protocol. For Research Use Only. Not for use in Diagnostic Procedures. 1 - 96 1 - 24 1 - 12 LV MV HV 10 µl - 400 µl 200 µl - 4 ml 1 ml - 10 ml Volume Configuration Qty. of Samples Sample Volumes chemagic Rod Head 96 24 12
- 3. HiSeq Ultra Rapid Sequencing / Bioinformatics Analysis Standard HiSeq 2500 instruments were customized to decrease overall sequencing time. Onboard clustering is utilized and completes in ~1.5 hours. Individual cycle times are ~6.5 minutes. Runs are maintained at 2x100, and standard v1 SBS reagents are used. For Research Use Only. Not for use in Diagnostic Procedures. The average sequencing run time was ~20 hours with bioinformatics analysis done in ~3.5 hours. The average amount of data was 62 GB; including two runs* (see tab. 2, run 617 and 624). with fluidics errors resulting in the loss of data from the entire bottom of the flow cell. Quality remained high throughout the run. Alignment and variant detection done with Illumina Isaac software v1, variant characterization and functional annotation done with CMH’s RUNES software. 602 606 621 633 604 617* 623 Run 19 hr 53 min 19 hr 43 min 23 hr 10 min 22 hr 17 min 21 hr 31 min 21 hr 8 min 19 hr 34 min 50.12 59.94 77.64 70.70 66.00 36.57 58.83 90.0 91.3 93.1 90.4 90.6 93.0 95.4 603 607 622 634 605 618 624* 19 hr 54 min 19 hr 45 min 23 hr 8 min 22 hr 19 min 21 hr 29 min 20 hr 52 min 19 hr 33 min 2 hr 12 min 2 hr 21 min 3 hr 22 min 2 hr 57 min 2 hr 30 min 2 hr 34 min 1 hr 57 min 29 min 31 min 39 min 30 min 34 min 29 min 24 min 30 min 29 min 41 min 36 min 30 min 36 min 37 min 3 hr 11 min 3 hr 21 min 4 hr 42 min 4 hr 3 min 3 hr 34 min 3 hr 39 min 2 hr 58 min 58.90 62.75 78.61 71.15 66.07 71.31 36.67 91.3 91.9 92.6 90.7 91.1 93.6 95.2 Time GB % > Q30 Isaac Alignment Variant Calling RUNES Total Bioinformatics Fig. 4: Screenshot, Illumina Sequencing Analysis Viewer. Tab. 2: Example run timings from 14 ultra rapid runs.
- 4. Ultra Rapid Protocol Fig. 5: Modified ultra rapid WGS. Modifications were made to all aspects of WGS. The largest decrease in time was accomplished by implementing Isaac for alignment. Run times were decreased ~5 hours by modifying the chemistries and cycle times of a standard HiSeq2500 in rapid mode. Further modest time savings in sample preparation were accomplished by using the KapaHyper library prep kit omitting the final PCR amplification step. In total, including DNA isolation WGS may be completed in <35 hours, including analysis. Sensitivity / Specificity Sensitivity and Specificity are calculated using NA12878, which has been highly characterized for SNPs and indels. With ~40x coverage from two flowcells in ultra rapid mode configuration, sensitivity and specificity were both >99%. Tab. 3: Calculation of sensitivity and specificity. Conclusion The chemagic MSM I (PerkinElmer) enables fast DNA isolation of high volume blood samples by coincidentally providing high quality DNA suitable for sequencing. Modifications to all aspects of Whole Genome Sequencing have led to a 40% decrease in the time. • Importantly, ultra rapid run mode does not affect run quality scores or variant detection sensitivity and specificity. • Further modifications will enable WGS to be completed in <24 hours [1]. Acknowledgements This work was supported by the Marion Merrell Dow Foundation, Children’s Mercy - Kansas City, W.T. Kemper Foundation, Pat & Gil Clements Foundation, Black & Veatch, and NICHD and NHGRI (U19HD077693). References 1. Miller NA, Farrow EG, Gibson M, Willig LK, Twist G, Yoo B, Marrs T, Corder S, Krivohlavek L, Walter A, Petrikin JE, Saunders CJ, Thiffault I, Soden SE, Smith LD, Dinwiddie DL, Herd S, Cakici JA, Catreux S, Ruehle M, Kingsmore SF. 2015. A 26-hour system of highly sensitive whole genome sequencing for emergency management of genetic diseases. Genome Med. Sep 30;7(1):100. Author Affiliations 1 Center for Pediatric Genomic Medicine, 2 Department of Pediatrics, and 3 Department of Pathology, Children‘s Mercy - Kansas City, Kansas City, Missouri 64108, USA 4 School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri 64108, USA • Consent Blood Sample 0 • DNA Isolation 1.5 • Sample QC/Dilution/Shearing 1 • Library Preparation 1.5 • Sample QC 1.5 • Sequencing Run 20 • Isaac Alignment 1 • Variant Calling 0.3 • RUNES 0.5 • Preliminary Results PerkinElmer chemagen Technologie GmbH Arnold-Sommerfeld-Ring 2 52499 Baesweiler, Germany Phone: +49 (0)2401 805 501 Fax: +49 (0)2401 805 519 info@chemagen.com, www.chemagen.com 2702149NA12878 - 633 + 634 7747 3216 479489 3181638 99.71% 99.33% True + False - False + True - Total Sens. Spec. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Phone: (+1) 800-762-4000 or (+1) 203-925-4602 www.perkinelmer.com CT72141601-01 Printed in Germany Copyright ©2016, PerkinElmer Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners.