Mar 25, 2020
- Graham J Etherington1,
- Darren Heavens1,
- David Baker1,
- Ashleigh Lister1,
- Rose McNelly1,
- Gonzalo Garcia1,
- Bernardo Clavijo1,
- Iain Macaulay1,
- Wilfried Haerty1,
- Federica Di Palma1
- 1The Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ, United Kingdom
- GigaScience Press
External link: https://doi.org/10.1093/gigascience/giaa045
Protocol Citation: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma 2020. Bionano genome mapping from animal tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bd7ei9je
Manuscript citation:
Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma, Sequencing smart: De novo sequencing and assembly approaches for a non-model mammal, GigaScience, Volume 9, Issue 5, May 2020, giaa045, https://doi.org/10.1093/gigascience/giaa045
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2020
Last Modified: March 26, 2020
Protocol Integer ID: 34758
Keywords: polecat, vertebrate, non-model organism, Illumina, chromium, Bionano, assembly, sequencing,
Abstract
Bionano genome map protocol
The Bionano genome maps were prepared by taking approximately 10 mg of DNA (in this case extracted from a polecat) from the sample stored in 100% EtOH.
The IrysPrep Animal Tissue DNA Isolation from Fibrous Tissue protocol was followed using the IrysPrep Animal Tissue DNA kit (RE-013-10) from Bionano Genomics.
The animal tissue sample was cut up into <3mm pieces for homogenisation and then fixed in a 2% formaldehyde solution in kit-provided Homogenisation Buffer (HB) for 00:30:00 On ice before blending using a Qiagen Tissueruptor.
Spin at 1500x g for 00:05:00 in centrifuge, remove supernatant and resuspend in around 50 µL of HB buffer using wide bore tip to produce a total volume of 66 µL .
40 µL of LMP agarose was then melted at 70 °C and cooled to 43 °C before addition to the cell resuspension and mixing using a wide bore tip.
One plug of around 90 µL was cast using the Chef Mammalian Genomic DNA Plug Kit (Bio-Rad 170-3591).
Once cooled to 4 °C the plug was added to a lysis solution containing 200 µL proteinase K (QIAGEN 158920) and 2.5 mL of Bionano lysis Buffer.
This was incubated at 50 °C for 02:00:00 in a thermomixer, making a fresh proteinase K solution and incubating Overnight .
The 50 mL tubes were then removed from the thermomixer for 00:05:00 before 50 µL RNAse A (Qiagen158924) was added and to the tubes, returned to the thermomixer for a further hour at 37 °C .
The plugs were then washed 7 times in the Wash Buffer supplied with the Chef kit and 7 times in 1xTE.
The plug was removed and melted for 00:02:00 at 70 °C followed by 00:05:00 at 43 °C before adding 10 µL of 0.2U/µl of GELase (Cambio Ltd G31200).
After 00:45:00 at 43 °C the melted plug was dialysed on a 0.1 uM membrane (Millipore VCWP04700) sitting in 15 mL of 1xTE buffer in a small petri dish.
After 00:45:00 the sample was removed with a wide bore tip and mixed gently 00:45:00 and left Overnight at 4 °C . A small amount was removed to QC on an Opgen Argus Q-Card and Qubit HS to calculate the DNA concentration.
300 ng of DNA was taken into the NLRS (Nick, Label, Repair and Stain) reaction using 1 µL Nt.BspQI (NEB R0644S). The optical maps were then generated using the Bionano Irys platform.