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MiSeq protocol and info

| 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.
| posted 31 Jan, 2022 20:33
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
| posted 31 Jan, 2022 20:43
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?
 
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