Rapid Nucleic Acid Isolation Method for Next-generation

Sequencing Applications Brown MT, Ferguson TM, Doebler R

Claremont BioSolutions LLC, Upland, CA

The introduction of the MinION sequencer has contributed significantly to advances in next generation

sequencing (NGS), providing researchers a field portable NGS tool capable of achieving ultra-long DNA reads. As

applications for the MinION continue to rapidly expand, one area that continues to be overlooked and

consequently lags behind is sample preparation.

In an effort to facilitate upstream nucleic acid extraction, Claremont BioSolutions (CBIO) has developed

novel sample preparation methods for the rapid isolation of DNA or RNA. Compact and field portable, the

technology can be used to isolate nucleic acids from difficult eukaryotes and prokaryotes present in complex

sample matrices (i.e. stool, sputum, blood, soil, and tissue) in as little as 15 minutes. Initial testing of DNA isolated

from E.coli and human pancreatic tumor tissue shows that DNA samples are compatible with the MinION and can

achieve read lengths >100 Kb. CBIO’s technology is customizable depending upon the application and can be

integrated into a cartridge format to allow for automated sample preparation solutions. Therefore, due to

customizability for a variety of bench and field applications, CBIO’s sample preparation methods appear well-

suited for use with the MinION sequencing platform.

Background

Conclusions

For applications that require ultra-long sequence reads, including the detection of structural variants and

antibiotic resistant islands, the MinION platform offers significant promise. In our preliminary testing we were able

to sequence ultra-long DNA (>100 Kb) from both E. coli and pancreatic tumor tissue samples purified using

DNAexpress™ technology and align the reads with reference genomes. The system was relatively

straightforward to setup and very mobile, which fits well with our applications. While we did observe significant

loss of ultra-long DNA during library preparation there appear to steps that can be employed to minimize loss

and or shearing. Overall, we felt the MinION sequencer offered a convenient and effective mobile solution for our

downstream NGS needs and validated our rapid method for ultra-long DNA isolation from bacteria and tissue

samples.

Funding for this poster was made possible in part by NIH Phase I SBIR Grant 5R43GM109502-2

Abstract

Figure 1. Rapid Sample Preparation

Results

Sample preparation

E. coli Cell Lysis

Method 1. 5x108 cfus were lysed by passing the cells through an OmniLyse®

device (Figure 3A) for two minutes using a 3 volt battery pack.

Method 2: 5x108 cfus were incubated in DNAexpress buffer containing 50 mg of

lysozyme for 10 min at 37oC.

Tissue Sample Preparation

Frozen mouse and human tissue (10mg) were thawed in DNAexpress buffer

and disaggregated using a microHomogenizer™ device (Figure 3B) for 2

minutes using a 1.5v volt battery pack.

Methodology

Figure 4. PFGE Analysis of Purified DNA

L = Lambda hindi marker (48Kb -1 Mb)

Lane 1. E. coli DNA isolated using DNAexpress™ kit following

lysis using OmniLyse

2. E. coli DNA isolated using DNAexpress™ column following

lysozyme digestion and prototype DNAexpress column

3. E. coli DNA isolated using DNAexpress™ kit following

lysozyme (no bead beating) and modified DNAexpress column

4. DNA isolated from tissue, post-microHomogenizer and modified

DNAexpress

5. DNA isolated from mouse lung, post-microHomogenizer and

modified DNAexpress

MinION Sequencing

• Nanopore sequencing technology is set to revolutionize the field of genomics by offering the advantage of

long-read sequencing in a rapid and cost effective platform. In contrast to 2nd generation short-read NGS

technology, nanopore technology allows long-read sequencing of genomic DNA

• Possible improvements in: (1) detection of cancer associated structural variants (Norris, 2015), (2)

characterization of emerging antibiotic resistance bacterial strains (Ashton, 2015; Karlsson, 2015), or (3)

adoption of NGS in clinical settings

• To achieve long-read sequencing, tools to rapidly and effectively isolate high molecular weight DNA for

downstream library preparation are needed

Claremont BioSolutions’ Sample Preparation (Figure 1-2)

• CBIO’s sample preparation technology offers a rapid upstream method to isolate nucleic acid that is

compatible with downstream PCR, isothermal amplification, and lateral flow detection

• Compact and battery powered, CBIO’s technology is adaptable to the bench, biological safety hood or field

applications, including space

• CBIO’s technology can be used to isolate total nucleic acid, RNA and DNA (including ultra-long DNA)

References

Ashton, P.M., et al. (2015). MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance

island. Nature Biotechnology, 33; 3, 296-300.

Frith, M.C., et al. (2010). Parameters for accurate genome alignment. BMC Bioinformatics, 11, 80.

Karlsson, E., et al. (2015). Scaffolding of a bacterial genome using MinION nanopore sequencing. Scientific Reports, 5.

Li, H., et al., The Sequence Alignment/Map format and SAMtools. Bioinformatics, 25; 16, 2078-2079.

Norris, A.L., et al. (2016). Nanopore sequencing detects structural variants in cancer. Cancer Biol Ther, 17; 3, 246-253.

Schonfeld, J. (2015) Quantitative PCR tools for spaceflight studies of gene expression aboard the International Space Station,

NASA Facts. FS-2015-06-01-ARC.

Watson, et al. (2015). poRe: an R package for the visualization and analysis of nanopore sequencing data. Bioinformatics, 31;1

114-115.

Figure 2. Integration and Automation of Sample Preparation

CBIO SimplePrep® Technology

Automated lysis and DNA

extraction from hard-to-lyse

samples in 6 minutes

NASA Ames WetLab-2 SPM

Integrates CBIO’s technology for rapid

extraction of RNA from biological

samples in space -currently on ISS to

isolate RNA for gene expression

studies (Schonfield, 2015)

E. coli Panc

Total reads 6,529 28,510

2D Workflow Success 847 2325

Total 2D Yield 21 Mb 36 Mb

Longest 2D 121,683 bp 445,574 bp*

Peak 2D Sc 9.9 9.7

Median 2D Sc 8.6 8.9

Figure 3. ClaremontBio’s (A) OmniLyse®

and (B) microHomogenizer™ Devices

DNA size was analyzed using pulsed-field gel electrophoresis (PFGE; Fig. 4) and linear stretching methods (data

not shown).

• DNA isolated using DNAexpress from OmniLyse-treated cells averaged ~40 Kb

• DNA isolated using DNAexpress from samples prepared using enzymatic lysis or homogenization was ultra-

long (avg. ~200 Kb)

OmniLyse

microHomogenizer

DNAexpress/RNAexpress

or PureLyse (3 min lysis/extraction)

MinION Library Prep

DNA from E. coli and human pancreatic tumor tissue were chosen for MinION analysis and libraries were

prepared with 1 µg of purified DNA. As per the MinION genomic library protocol, the DNA was first treated

using the an FFPE repair kit (NEB). All steps of the genomic DNA sequencing protocol were followed with the

exception of the Covaris g-tube shearing step in order to to keep the DNA ultra-long. Wide bore tips we used

in the preparation of the pancreatic tissue library to minimize DNA shearing.

MinION Run and Data Analysis

The MinION R7 flow cells were run for >36 hours on MinKNOW software and base calling was performed

using Metrichor™software (Oxford Nanopore). Fast5 files were converted to fastq and fasta files, and

histogram analysis was performed using poRe (Watson, 2014). Reads were aligned to reference sequences

using LAST (Frith, 2010) and converted to BAM format using maf-convert and SAMtools (Li, 2009).

DNA isolation and Analysis

Cell lysates, described above, were mixed with DNAexpress binding buffer and DNA was loaded onto the

CBIO’s fast flow DNAexpress™ column. Bound DNA was washed twice and the DNA was eluted in TE

buffer as per the kit protocol. Purified DNA was quantified using a Tecan m200 pro Nanoquant instrument.

DNA size was analyzed using pulse field gel electrophoresis as shown in Figure 4.

Table 1. Metrichor Data Output

Isolated DNA from E. coli and human pancreatic tumor tissue

was prepared using the genomic library protocol. We observed

a loss of 80-90% of the ultra-long DNA during the library prep

(DNA was recovered from AMP Pure beads after library prep).

The final yield was 197ng of DNA for the E. coli sample and 72

ng for the pancreatic sample.

During the sequencing runs, MinKNOW reported 139 active

pores in the E. coli flow cell and 995 active pores in the

pancreatic tissue flow cell. Both flow cells were run for >36

hours. We observed no issues during the E. coli DNA run but

MinKNOW reported “No data received” errors during the

pancreatic DNA run. We stopped the run, rebooted the system

and restarted MinKNOW. The events happened twice and

after restarting we were able to continue to collect data,

including ultra-long DNA sequence reads.

A B

Figure 5. MinION Run Data Analysis. 2D Histogram analysis for E. coli (A) and Human Pancreatic Tumor Tissue

(C) 2D “pass” data sets. Plot of Read Length vs. Quality Score for E. coli (B) and pancreatic tumor tissue (D). 2D

reads are plotted in red (qscore >9) and green (qscore <9), and 1D reads are plotted in blue.

Figure 6. Alignment of E. coli and Pancreatic Tissue

Reads with reference sequence. (A) Blastn analysis of

an ultra 2D long read from E. coli (102 Kb) showing 83%

alignment with reference K12 E. coli genome. (B)

Alignment of the E. coli 2D data set with the K-12 genome

generated 90% coverage (2X) in NCBI Genome

Workbench. (C) Blastn analysis of a long read 1D

sequence (86 Kb) from the pancreatic tissue data set

showed alignment with a gene for SLIT-ROBO Rho

GTPase Activating Protein 2C from Human chromosome 1.

The query sequence contained a possible 18 Kb insert

when aligned with the genome.

MinION Sequencing and Data Analysis

Sizing of DNAexpress™ kit purified DNA

From the Metrichor analysis both the E. coli and pancreatic MinION runs generated 2D and 1D reads >100 Kb

(Figure 5 A-D). The mean size of reads was 10 Kb for the E. coli sample and 4 Kb for the pancreatic sample,

which was significantly less that observed in the PFGE analysis. The difference in mean read length may be

due to shearing during library preparation or loss of larger DNA (entanglement with beads). 2D reads for both

data sets aligned with ~80-85% identity with reference genomes while template read alignments were lower

than 80% identity.

A C

B C

*Panc Longest 2D read of 445 Kb appears to be

the addition of the template (276 Kb) and

complement strand (207 Kb)

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