SEA-PHAGES | MiSeq protocol and info

Link to this post \| posted 26 Jan, 2022 17:41
uOttawaPHAGE	We will be able to do a MiSeq run later this term, but we need to prepare the library and purchase the reagents. Ideally we'd like to do this the same way it is done at Pitt, so this is largely directed at Dan and Becky, but anyone who has done MiSeq sequencing, please feel free to comment too. 1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads? 2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY? 3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money. 4. Do you use the Illumina v2 reagent kit? 5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit? 6. I believe Pitt uses a Qbit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings? 7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….). 8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached. 498Kb

Link to this post | posted 26 Jan, 2022 17:41

We will be able to do a MiSeq run later this term, but we need to prepare the library and purchase the reagents. Ideally we'd like to do this the same way it is done at Pitt, so this is largely directed at Dan and Becky, but anyone who has done MiSeq sequencing, please feel free to comment too.

1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads?

2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY?

3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money.

4. Do you use the Illumina v2 reagent kit?

5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit?

6. I believe Pitt uses a Qbit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings?

7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….).

8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached.

Link to this post \| posted 31 Jan, 2022 20:33
DanRussell	Hi Adam, Some answers below. 1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads? Yes, that's one. Any of the NEBNext Ultra II FS kits will work. The "FS" part is for shearing your DNA. 2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY? When we do our typical runs of 48 phages, we use dual indexing with this barcode kit (#E7600S): https://www.neb.com/products/e7600-nebnext-multiplex-oligos-for-illumina-dual-index-primers-set-1#Product%20Information That one gives 96 possible dual-indexed combos. When we do smaller runs, we sometimes use single-indexing with a kit like this (#E7335S): https://www.neb.com/products/e7335-nebnext-multiplex-oligos-for-illumina-index-primers-set-1 You can do it DIY, some people do, but not us! 3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money. We've never tried, but FYI our output library concentration from the current workflow is often not too far above the "minimum" 4nM required for proper MiSeq loading, so it's possible you'd be too low. Also, some of the volumes would be quite low, like resuspending beads in 8.5 µl of buffer, which could be a bit tricky. 4. Do you use the Illumina v2 reagent kit? We use 150-cycle v3 Illumina kits. You can instruct the MiSeq to do 1x150bp reads. (Rather than paired end reads.) 5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit? Check the NEB and Illumina protocols for specifics about what's needed, but there's no too much that's not standard lab fare. Washing the MiSeq is just done with Tween diluted in water, so you'll need the right Tween, and water, but no kit necessary. 6. I believe Pitt uses a Qubit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings? Yes, you'll need a Qubit Broad Range DNA Kit to quantify DNA using a Qubit. We use 1 µl of sample and 199 µl of QWS. You then just have to tell the Qubit itself what you used, and it'll tell you your stock concentration. 7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….). As mentioned above, we use 150-cycle v3 kits, set to 1x150bp reads. When making a sample sheet, we select "FASTQ only". 8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached. Definitely haven't done that. You can get the above-referenced library prep kits with beads included, but I think basically any of the purification beads are okay as long as they work. In general, it sounds like you're trying to keep library prep costs low which is understandable. Perhaps even better than half-volume reactions would be to combine two phages you know—for example, by restriction digest—belong to different clusters. Then you'd get two complete phage sequences out of each library prep. There's no doubt that people use a variety of DIY and tweaks, but we've found that the cost per library from NEB is < $40 and we're more concerned with throughput and success %, so we haven't experimented. Good luck! –Dan Edited 31 Jan, 2022 20:35

Link to this post | posted 31 Jan, 2022 20:33

DanRussell

Hi Adam,

Some answers below.

1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads?

Yes, that's one. Any of the NEBNext Ultra II FS kits will work. The "FS" part is for shearing your DNA.

2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY?

When we do our typical runs of 48 phages, we use dual indexing with this barcode kit (#E7600S): https://www.neb.com/products/e7600-nebnext-multiplex-oligos-for-illumina-dual-index-primers-set-1#Product%20Information

That one gives 96 possible dual-indexed combos. When we do smaller runs, we sometimes use single-indexing with a kit like this (#E7335S): https://www.neb.com/products/e7335-nebnext-multiplex-oligos-for-illumina-index-primers-set-1

You can do it DIY, some people do, but not us!

3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money.

We've never tried, but FYI our output library concentration from the current workflow is often not too far above the "minimum" 4nM required for proper MiSeq loading, so it's possible you'd be too low. Also, some of the volumes would be quite low, like resuspending beads in 8.5 µl of buffer, which could be a bit tricky.

4. Do you use the Illumina v2 reagent kit?

We use 150-cycle v3 Illumina kits. You can instruct the MiSeq to do 1x150bp reads. (Rather than paired end reads.)

5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit?

Check the NEB and Illumina protocols for specifics about what's needed, but there's no too much that's not standard lab fare. Washing the MiSeq is just done with Tween diluted in water, so you'll need the right Tween, and water, but no kit necessary.

6. I believe Pitt uses a Qubit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings?

Yes, you'll need a Qubit Broad Range DNA Kit to quantify DNA using a Qubit. We use 1 µl of sample and 199 µl of QWS. You then just have to tell the Qubit itself what you used, and it'll tell you your stock concentration.

7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….).

As mentioned above, we use 150-cycle v3 kits, set to 1x150bp reads. When making a sample sheet, we select "FASTQ only".

8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached.

Definitely haven't done that. You can get the above-referenced library prep kits with beads included, but I think basically any of the purification beads are okay as long as they work.

In general, it sounds like you're trying to keep library prep costs low which is understandable. Perhaps even better than half-volume reactions would be to combine two phages you know—for example, by restriction digest—belong to different clusters. Then you'd get two complete phage sequences out of each library prep.

There's no doubt that people use a variety of DIY and tweaks, but we've found that the cost per library from NEB is < $40 and we're more concerned with throughput and success %, so we haven't experimented.

Good luck!
–Dan

Edited 31 Jan, 2022 20:35

Link to this post \| posted 31 Jan, 2022 20:43
uOttawaPHAGE	thanks Dan. The library cost is clearly what adds up, which is why the 1/2 vol question. We will probably try that for a reaction or two. Sounds like you use the NEB beads?

Link to this post \| posted 27 May, 2022 15:49
nietof	Hi, We just got a MiniSeq and plan to try our own sequencing. I would like to follow your protocol. Would you be able to share the protocol you use for sequencing? Any recommendations for the MiniSeq? Recommended coverage? Any information is greatly appreciated. regards, Fernando Edited 27 May, 2022 17:15

Link to this post \| posted 27 May, 2022 17:22
DanRussell	Hi Fernando, I'd recommend shooting for 100X coverage on your phage genomes. It might be a little higher or lower, but that's generally a decent target that's also not going to cost too much per phage. If you take an "average" phage genome of 70kb in length, that means you need ~7,000,000 bp of sequence to get to that coverage level. If the reads you're doing are 150 bases long, then: 7,000,000/150 = ~47,000 reads per genome Obviously that'll change depending on the size of the genome (often unknown!) and the length of the reads, so we usually shoot for ~100,000 reads per phage genome, though 50,000 will often be enough. As for protocols, we just use the standard protocol that comes with our library prep kit: https://www.neb.com/nebnext-ultra-ii-fns-dna/nebnext-ultra-ii-for-dna-library-prep After library prep, we use BioAnalyzer HS chips to check quantity/quality. Then the standard Illumina protocol to load the sequencer. We have a MiSeq, not a MiniSeq, so I don't have any specifics for the MiniSeq, but most of Illumina's sequencers these days are pretty easy to use. Take care and good luck! –Dan

Link to this post | posted 27 May, 2022 17:22

DanRussell

Hi Fernando,

I'd recommend shooting for 100X coverage on your phage genomes. It might be a little higher or lower, but that's generally a decent target that's also not going to cost too much per phage.

If you take an "average" phage genome of 70kb in length, that means you need ~7,000,000 bp of sequence to get to that coverage level. If the reads you're doing are 150 bases long, then:

7,000,000/150 = ~47,000 reads per genome

Obviously that'll change depending on the size of the genome (often unknown!) and the length of the reads, so we usually shoot for ~100,000 reads per phage genome, though 50,000 will often be enough.

As for protocols, we just use the standard protocol that comes with our library prep kit:
https://www.neb.com/nebnext-ultra-ii-fns-dna/nebnext-ultra-ii-for-dna-library-prep

After library prep, we use BioAnalyzer HS chips to check quantity/quality.

Then the standard Illumina protocol to load the sequencer.

We have a MiSeq, not a MiniSeq, so I don't have any specifics for the MiniSeq, but most of Illumina's sequencers these days are pretty easy to use.

Take care and good luck!
–Dan

Link to this post \| posted 27 May, 2022 22:24
nietof	Dan Muchas gracias!!! Regards Fernando

Link to this post \| posted 02 Jun, 2022 15:20
nietof	Dan One last question, are you using the 300-cycle kit at 2x150 paired end reads that you are running on the MiSeq? Thank you Fernando

Link to this post \| posted 01 Aug, 2022 12:55
uOttawaPHAGE	Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 01 Aug, 2022 12:55

uOttawaPHAGE

Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions:
1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence?
2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome.
thanks again for your help with these questions.
Adam

Link to this post \| posted 01 Aug, 2022 12:58
uOttawaPHAGE	Hi Dan. We are finally making our libraries and I was curious if you use the Bioanalyzer to check the library - based on the question above from Fernando it sounds like you do. Some questions: 1. I've not worked with data off the bioanalyzer - is there a cut-off for how you determine if a library is good enough to sequence? 2. Do you use the Qubit before or after library prep? for some reason I thought you used it after, but I'm thinking I have that wrong and you use it before to assess the quality/quantity of the genome. thanks again for your help with these questions. Adam

Link to this post | posted 01 Aug, 2022 12:58

uOttawaPHAGE

Link to this post \| posted 02 Nov, 2022 20:30
uOttawaPHAGE	Hi Dan. We're finally about to do our inaugural MiSeq run with ~20 libraries. A few final questions, if you have time: 1. We were going to use the Illumina Protocol A for denaturing/loading - I'm assuming using Bioanalyzer results constitutes "standard normalization"? 2. Do you denature 5uL of 4nM pooled libraries? 3. And to clarify, with 20 libraries each at 4nM, that would only be 0.25uL of each library. 4. IN a response above you mentioned your libraries are often not much above 4nM. Ours are higher, so I'm wondering if we did our library prep differently. We used the NEBNext Ultra II FS kit. How much input genome to you use? 5. Do you add 1% PhiX as a control? This seems standard. 6. Any other subtleties I should know about? thanks again for your help with these questions. Adam

Link to this post | posted 02 Nov, 2022 20:30

uOttawaPHAGE

Hi Dan. We're finally about to do our inaugural MiSeq run with ~20 libraries. A few final questions, if you have time:
1. We were going to use the Illumina Protocol A for denaturing/loading - I'm assuming using Bioanalyzer results constitutes "standard normalization"?
2. Do you denature 5uL of 4nM pooled libraries?
3. And to clarify, with 20 libraries each at 4nM, that would only be 0.25uL of each library.
4. IN a response above you mentioned your libraries are often not much above 4nM. Ours are higher, so I'm wondering if we did our library prep differently. We used the NEBNext Ultra II FS kit. How much input genome to you use?
5. Do you add 1% PhiX as a control? This seems standard.
6. Any other subtleties I should know about?
thanks again for your help with these questions.
Adam

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