Sequencing library preparation for MinIONTM and … · Sequencing library preparation for MinION TM...
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Sequencing library preparation for MinION TM and PromethION TM Several gDNA library-prep protocols are available, offering low DNA input, rapid reaction and ultra-long reads Fig. 1 Schematic representation of ligation-based 2D library preparation We have developed workflows to maximise throughput from both high- and low-input samples, without the need for PCR (Figs. 1a and 1b). Omitting the PCR step means that amplification bias is eliminated and several hours of laboratory time are saved. The PCR-free low-input protocol is compatible with longer fragments than the PCR-based protocol, giving the user longer reads. Using the low-input protocol, the user loads the library into the MinION while strands are still bound to the beads. Gravity draws the beads onto the membrane surface, increasing the local concentration of DNA molecules on the membrane. For applications where the accuracy of a 1D read (template strand only) is of sufficient quality for the downstream analysis, we have developed a rapid library prep. This prep uses a transposase enzyme, which simultaneously fragments and attaches adapters to double-stranded DNA. Starting with a DNA extract, the entire library prep can be performed in approximately 5 minutes (Fig. 2). In spite of the obligatory fragmentation step, this library prep is capable of generating extremely long reads. We are currently developing a 2D version of this library prep, which will take approximately 30 minutes to get from gDNA to the finished library. Although all of our gDNA library preparations are compatible with reads of several kilobases in length, there are some applications for which it would be preferable to have even longer reads, such as de novo assembly of complex genomes, or phasing of SNPs over long distances. To address this need, we have developed a workflow which is capable of depleting shorter fragments from a library, leaving only the long fragments (Fig. 3). It is necessary to begin the library prep with high molecular weight DNA and to take precautions to avoid shearing the strands during preparation. In this workflow the library is prepared using any of the PCR-free protocols, being careful not to shear the DNA, and the adapter-ligated library is then size-selected on a BluePippin. Figs. 4a and 4b show a library prepared using the standard ligation-based 2D protocol before and after size selection. Depending on the fragment size in the original gDNA sample, this protocol is capable of generating extremely long reads. Fig. 4c shows a read, obtained from an E. coli gDNA extract, in which the template and complement strands are each 255 kb, giving an overall sequence length of 510 kb. Nanopore sequencing 2D library prep and low-input protocol Rapid 1D and 2D library preps Ultra-long reads using Blue Pippin size selection Size selection removes fragments below a specified length Fig. 3 Ultra-long-read workflow Fig. 2 Schematic representation of rapid 1D and 2D protocols Fig. 4 Ultra-long reads from the Blue Pippin workflow Contact: [email protected] More information at: www.nanoporetech.com and publications.nanoporetech.com © 2016 Oxford Nanopore Technologies. All rights reserved. Version 1 A A p p T T high molecular weight gDNA optional fragmentation adapter ligation tether attachment strep-bead selection elute and run standard, 250-1000 ng 70 minutes + end-prep Read length (kb) Events (10 6 ) Run time (hours) 280 240 200 160 120 80 40 0 0 run with beads low-input, 20 ng 1 2 3 4 5 6 a) b) 1000 ng (standard protocol) 20 ng (low-input protocol) high molecular weight gDNA transposome complex (transposase + adapters) simultaneous cleavage and addition of transposase adapters attachment of 1D sequencing adapters flap-sealing and ligation of 2D sequencing adapters Genomic DNA Nanopore library prep MinION sequencing Size selection Read length (kb) 0 20 40 60 80 100 0 2 4 6 8 10 12 14 No size-selection Bases sequenced (Mb) Read length (kb) 0 20 40 60 80 100 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 With size-selection Bases sequenced (Mb) Current (pA) 200 50 100 150 Time (s) 7387 1900 3000 4000 5000 6000 7000 255 kb template strand 255 kb complement strand hairpin a) b) c) 5 min 30 min
Transcript of Sequencing library preparation for MinIONTM and … · Sequencing library preparation for MinION TM...
Sequencing library preparation for MinIONTM and PromethIONTM
Several gDNA library-prep protocols are available, offering low DNA input, rapid reaction and ultra-long reads
Fig. 1 Schematic representation of ligation-based 2D library preparation
We have developed workflows to maximise throughput from both high- and low-input samples, without the need for PCR (Figs. 1a and 1b). Omitting the PCR step means that amplification bias is eliminated and several hours of laboratory time are saved. The PCR-free low-input protocol is compatible with longer fragments than the PCR-based protocol, giving the user longer reads. Using the low-input protocol, the user loads the library into the MinION while strands are still bound to the beads. Gravity draws the beads onto the membrane surface, increasing the local concentration of DNA molecules on the membrane.
For applications where the accuracy of a 1D read (template strand only) is of sufficient quality for the downstream analysis, we have developed a rapid library prep. This prep uses a transposase enzyme, which simultaneously fragments and attaches adapters to double-stranded DNA. Starting with a DNA extract, the entire library prep can be performed in approximately 5 minutes (Fig. 2). In spite of the obligatory fragmentation step, this library prep is capable of generating extremely long reads. We are currently developing a 2D version of this library prep, which will take approximately 30 minutes to get from gDNA to the finished library.
Although all of our gDNA library preparations are compatible with reads of several kilobases in length, there are some applications for which it would be preferable to have even longer reads, such as de novo assembly of complex genomes, or phasing of SNPs over long distances. To address this need, we have developed a workflow which is capable of depleting shorter fragments from a library, leaving only the long fragments (Fig. 3). It is necessary to begin the library prep with high molecular weight DNA and to take precautions to avoid shearing the strands during preparation.
In this workflow the library is prepared using any of the PCR-free protocols, being careful not to shear the DNA, and the adapter-ligated library is then size-selected on a BluePippin. Figs. 4a and 4b show a library prepared using the standard ligation-based 2D protocol before and after size selection. Depending on the fragment size in the original gDNA sample, this protocol is capable of generating extremely long reads. Fig. 4c shows a read, obtained from an E. coli gDNA extract, in which the template and complement strands are each 255 kb, giving an overall sequence length of 510 kb.
Nanopore sequencing 2D library prep and low-input protocol
Rapid 1D and 2D library preps
Ultra-long reads using Blue Pippin size selection
Size selection removes fragments below a specified length
Fig. 3 Ultra-long-read workflow
Fig. 2 Schematic representation of rapid 1D and 2D protocols
Fig. 4 Ultra-long reads from the Blue Pippin workflow
Contact: [email protected] More information at: www.nanoporetech.com and publications.nanoporetech.com
© 2016 Oxford Nanopore Technologies. All rights reserved.Version 1
A
Ap
p
TT
high molecular weight gDNA
optional fragmentation
adapter ligation
tether attachment
strep-bead selection
elute and run standard, 250-1000 ng
70 minutes
+
end-prep
Read length (kb)
Eve
nts
(106 )
Run time (hours)
280
240
200
160
120
80
40
00run with beads
low-input, 20 ng1 2 3 4 5 6
a) b)1000 ng (standard protocol)
20 ng (low-input protocol)
high molecular weight gDNA
transposome complex (transposase + adapters)
simultaneous cleavage and addition of transposase adapters
attachment of 1D sequencing adapters
flap-sealing and ligation of 2D sequencing adapters
Genomic DNA
Nanopore library prep
MinIONsequencing
Size selection
Read length (kb)0 20 40 60 80 100
0
2
4
6
8
10
12
14No size-selection
Bas
es s
eque
nced
(Mb)
Read length (kb)0 20 40 60 80 100
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
With size-selection
Bas
es s
eque
nced
(Mb)
Cur
rent
(pA)
200
50
100
150
Time (s)73871900 3000 4000 5000 6000 7000
255 kb template strand 255 kb complement strand
hairpin
a) b)
c)
5 min 30 min
