library prep using sonicator - indexing

[kerryp]

I'm currently using Nextera XT to prepare libraries for MiSeq. This involves 'tagmentation' which simultaneously fragments (enzymatically) and adds a tag sequence. We then amplify using Nextera XT indices.

My question is - if we were to fragment our dsDNA by sonication, how would we then amplify the fragments using Nextera XT indices? I assume the tag sequence is essential - is there any way around this?

Thanks in advance for any advice!

ps I initially posted this under 'illumina/solexa', apologies for double-posting!

[nucacidhunter]

You will need to ligate adapters similar to Nextera tags to be able to amplify them with Nextera primers if you see any advantage.

[kerplunk412]

It sounds like you just need a standard ligation-based DNA-Seq kit. Be aware that there are many available from many different companies, and you can save money and often get a better kit by going with someone other than Illumina.

[kerryp]

Thanks nucacidhunter and kerplunk412 - is there any particular kit you'd recommend?

I can see us moving away from Nextera as we don't use their normalisation method any more (so we're just using half the reagents in the kit anyway). But would like to avoid spending the next year optimising our runs!

[nucacidhunter]

That depends on the amount of DNA that is available. If you want to replace XT with another kit with low input, it would cost more. If you give more info on application and DNA amount it would be easier to narrow kits down.

[kerryp]

We're doing small genome resequencing of bacterial genomes. We're not restricted to low inputs - we could use more DNA if needed

[nucacidhunter]

In that case you can shop for price. Major brands to consider:
1. NEB
2. Bioo Scientific
3. KAPA
4. Illumina

Each will have few products and their sales department can help you to choose the right product in right price.

[kerplunk412]

If you can get "unlimited" amounts of input DNA a PCR-free kit is your best option. Bioo Scientific offers one that you can find here. Feel free to PM me for more information. As a disclaimer, I work for Bioo Scientific.