Sequencing several 1-2Mb bacterial genomes

apredeus · 12-27-2016, 09:55 AM

Hello all,

I was wondering what is the best protocol to sequence several bacterial genomes (1-2Mb in size) using the newest flowcells (R9) and and a MinION sequencer.

We want to assemble them de novo; if need be, we can add some Illumina reads as well.

It seems like ligation protocol 1D should give the best yield. I am struggling in finding the exact protocol with times etc. If you could point me to protocols that work best on the newest flowcells I'd be most grateful.

I was also wondering what is a good strategy for sequencing several genomes in 1 run - since it seems an overkill to run only one per flowcell. I know you can turn it off, but are there any good pooling strategies? Just adding things as we go on sequencing?

gringer · 12-28-2016, 02:10 AM

Either barcoding, or using a wash kit (in "storage" mode) should give reasonable results. Barcoding is the "safest" method, in terms of getting maximum yield out of the flow cell.

However, if you're sequencing sufficiently different bacterial genomes, it might work just throwing everything in together and getting Canu to sort out the contigs.

apredeus · 12-28-2016, 12:23 PM

Thank you. So for 1D regular genomic DNA you need this, right?

https://store.nanoporetech.com/catal...d/category/17/

Do you have to store both the sequencing kit and the barcodes at -20?

How many reactions does a standard 1D kit allow? The description says 6 but other users say it could be stretched to 10, does that sound right?

Sorry for the barrage of questions, I'm still not approved at Minion community forum but want to plan the experiment thoroughly.

gringer · 12-30-2016, 11:23 AM

Yes, that kit will work, but only with the 1D ligation kit. Both kits should be stored at -20.

Kits can be stretched, depending on how brave you want to be with adding different quantities of the adapter sequences. With the recommended amount, I haven't been able to get more than about 1 more preparation out of a kit.

apredeus · 01-04-2017, 08:16 AM

Quote:

Originally Posted by gringer

Yes, that kit will work, but only with the 1D ligation kit. Both kits should be stored at -20.

Kits can be stretched, depending on how brave you want to be with adding different quantities of the adapter sequences. With the recommended amount, I haven't been able to get more than about 1 more preparation out of a kit.

Thank you for the answers, I appreciate it.

I'm checking the storage conditions and it seems like currently there are no kits that could be stored at RT, rapid kit included. Is that right? So do all the people that do the sequencing in the field take the -20 fridges with them? Seems strange.

gringer · 01-04-2017, 10:52 AM

If sequencing in the field as a day trip, a small polystyrene box is sufficient for kit storage (or kit transport). The kits don't take up much space and can handle a fair amount of time at a higher temperature.

If the field sequencing is for longer than that, then taking a freezer would be a good idea at the moment. ONT is currently working on improvements in the kits so that they can handle RT storage.

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12-27-2016, 09:55 AM	#1
apredeus Senior Member Location: Bioinformatics Institute, SPb Join Date: Jul 2012 Posts: 151	Sequencing several 1-2Mb bacterial genomes Hello all, I was wondering what is the best protocol to sequence several bacterial genomes (1-2Mb in size) using the newest flowcells (R9) and and a MinION sequencer. We want to assemble them de novo; if need be, we can add some Illumina reads as well. It seems like ligation protocol 1D should give the best yield. I am struggling in finding the exact protocol with times etc. If you could point me to protocols that work best on the newest flowcells I'd be most grateful. I was also wondering what is a good strategy for sequencing several genomes in 1 run - since it seems an overkill to run only one per flowcell. I know you can turn it off, but are there any good pooling strategies? Just adding things as we go on sequencing?

12-28-2016, 02:10 AM	#2
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	Either barcoding, or using a wash kit (in "storage" mode) should give reasonable results. Barcoding is the "safest" method, in terms of getting maximum yield out of the flow cell. However, if you're sequencing sufficiently different bacterial genomes, it might work just throwing everything in together and getting Canu to sort out the contigs.

12-28-2016, 12:23 PM	#3
apredeus Senior Member Location: Bioinformatics Institute, SPb Join Date: Jul 2012 Posts: 151	Thank you. So for 1D regular genomic DNA you need this, right? https://store.nanoporetech.com/catal...d/category/17/ Do you have to store both the sequencing kit and the barcodes at -20? How many reactions does a standard 1D kit allow? The description says 6 but other users say it could be stretched to 10, does that sound right? Sorry for the barrage of questions, I'm still not approved at Minion community forum but want to plan the experiment thoroughly.

12-30-2016, 11:23 AM	#4
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	Yes, that kit will work, but only with the 1D ligation kit. Both kits should be stored at -20. Kits can be stretched, depending on how brave you want to be with adding different quantities of the adapter sequences. With the recommended amount, I haven't been able to get more than about 1 more preparation out of a kit.

01-04-2017, 10:52 AM	#6
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	If sequencing in the field as a day trip, a small polystyrene box is sufficient for kit storage (or kit transport). The kits don't take up much space and can handle a fair amount of time at a higher temperature. If the field sequencing is for longer than that, then taking a freezer would be a good idea at the moment. ONT is currently working on improvements in the kits so that they can handle RT storage.