SEA-PHAGES | Nanopore sequencing questions

Link to this post \| posted 04 Feb, 2022 21:02
uOttawaPHAGE	We may be purchasing a MINion nanopore sequencer and I was looking for details on what we'll need. I know Dan has done some Nanopore sequencing, but I assume some others have as well. Any suggestions would be great. Some questions: 1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information E7180S (24 rxns) 2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore: https://store.nanoporetech.com/ligation-sequencing-kit.html 3. And these barcodes for up to 24 samples: https://store.nanoporetech.com/native-barcoding-expansion-1-12.html https://store.nanoporetech.com/native-barcoding-expansion-13-24.html 4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume? 5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready…. 6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Link to this post | posted 04 Feb, 2022 21:02

We may be purchasing a MINion nanopore sequencer and I was looking for details on what we'll need. I know Dan has done some Nanopore sequencing, but I assume some others have as well. Any suggestions would be great. Some questions:

1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information
E7180S (24 rxns)

2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore:

https://store.nanoporetech.com/ligation-sequencing-kit.html

3. And these barcodes for up to 24 samples:

https://store.nanoporetech.com/native-barcoding-expansion-1-12.html

https://store.nanoporetech.com/native-barcoding-expansion-13-24.html

4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume?

5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready….

6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Link to this post \| posted 01 Mar, 2022 17:34
DanRussell	uOttawaPHAGE 1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information E7180S (24 rxns) Yes, we use that kit as called for in the protocol. uOttawaPHAGE 2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore: https://store.nanoporetech.com/ligation-sequencing-kit.html 3. And these barcodes for up to 24 samples: https://store.nanoporetech.com/native-barcoding-expansion-1-12.html https://store.nanoporetech.com/native-barcoding-expansion-13-24.html Yep, those are the ones we use though we've never done more than 8 at a time. uOttawaPHAGE 4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume? Haven't tried this. uOttawaPHAGE 5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready…. I honestly don't remember re: the starter pack, but in general you can ask for delayed ship dates on the Nanopore website if you don't want them to ship immediately. uOttawaPHAGE 6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course. Yes, flow cells sort of have a "useful lifetime" in hours of sequencing. They usually say the flowcells can sequence for 72 hours. So you can stop after 4 hours, wash, and then do a different run later. But there's a substantial decline over time (both active sequencing time and sedentary fridge time)in the number of available pores, so you might have to sequence the second run for 12 hours to get the same amount of sequence as the first run got in 4 hours. A third run might need 48 hours. And so on. So you can't really look at that "72 hours" as like 6 equal 12-hour runs. There's also some carryover in barcodes from one run to the next. Though it's not a ton, it can be confounding if you don't have a ton of reads. Good luck! –Dan

Link to this post | posted 01 Mar, 2022 17:34

DanRussell

uOttawaPHAGE
1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information
E7180S (24 rxns)

Yes, we use that kit as called for in the protocol.

uOttawaPHAGE
2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore:

https://store.nanoporetech.com/ligation-sequencing-kit.html

3. And these barcodes for up to 24 samples:

https://store.nanoporetech.com/native-barcoding-expansion-1-12.html

https://store.nanoporetech.com/native-barcoding-expansion-13-24.html

Yep, those are the ones we use though we've never done more than 8 at a time.

uOttawaPHAGE
4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume?

Haven't tried this.

uOttawaPHAGE
5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready….

I honestly don't remember re: the starter pack, but in general you can ask for delayed ship dates on the Nanopore website if you don't want them to ship immediately.

uOttawaPHAGE
6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Yes, flow cells sort of have a "useful lifetime" in hours of sequencing. They usually say the flowcells can sequence for 72 hours. So you can stop after 4 hours, wash, and then do a different run later. But there's a substantial decline over time (both active sequencing time and sedentary fridge time)in the number of available pores, so you might have to sequence the second run for 12 hours to get the same amount of sequence as the first run got in 4 hours. A third run might need 48 hours. And so on. So you can't really look at that "72 hours" as like 6 equal 12-hour runs.

There's also some carryover in barcodes from one run to the next. Though it's not a ton, it can be confounding if you don't have a ton of reads.

Good luck!
–Dan

Link to this post \| posted 01 Mar, 2022 17:47
uOttawaPHAGE	thanks Dan! I'll keep you updated on our efforts. We did find out we have a year to get the flow cell - so we can wait until our first libraries are ready.

Recent Activity

Nanopore sequencing questions