I am doing a research stay at an institute and they offered to resequence the genome of one of my strains. We have the pair end sequences but the assembly went not so good so they suggested resequencing with the mate pair kit.
Nobody has experience and there is trouble with the protocol, as most of the DNA is lost during library prep. They are using the alternative protocol for cleaning up after strand displacement, because is the kit they have.
Any tip or suggestion will be welcome. Thanks!
I do not really have experience with the Mate Pair prep kit, but there should be alternative solutions for your problem:
1) Long-read sequencing (PacBio, Oxford Nanopores)
There should be people offering this as a service (try this: http://allseq.com/)
2) Virtual Long read sequencing
For Illumina Sequencing you have the possibility to create "virtual long reads", which use a barcoding system to detect which initial DNA fragment the read derived from (https://www.10xgenomics.com/).
Both of these methods should enhance your assembly.
Best,
Peter
Thank you Peter. but the kit was already bought and it's expensive and the illumina MiSeq machine is already at the institute. So there should be a way to use the resources we already have paid for. hope there is!
Hi. I think you made a right, affordable choice. We work on vertebrates, mainly on HiSeq, but have a plenty of experience of using the illumina Nextera Mate Pair Lib Prep kit. Please refer to our publication and protocol guide available online, first: http://www2.clst.riken.jp/phylo/imate.html
Please let me know if you have further questions, etc. I can guide step-by-step if it can help your study.
Shigehiro
Unit Leader
Phyloinformatics Unit, RIKEN Center for Life Science Technologies (CLST)
http://www.clst.riken.jp/phylo/
Dear Shigehiro
Thank you very much!!! I will look at your protocol and when we try again I'll let you know how it worked and if it doesn't I'll accept your offer of guidance.
Best regards,
Susana
As I stated earlier, we work on large vertebrate genomes. So, just doing as we do may not be optimal for your purpose. Please be careful with this. Plus, you don't have to fragment DNA with Covaris too much, if you will get long reads on MiSeq. Several factors (like fragmentation extent, read length, and the way you use the resultant reads) are interconnected and relative to each other. Our protocol guide should tell this complicated logic to you rather than concrete reaction conditions, etc.
Shigehiro
Hey Susana,
I have done a lot of mate pair libraries! The MiSeq will work just fine no problems there.
There could be a couple of reasons why you lost most of the DNA.
1) I would strongly recommend to use the Zymo kit for clean up and concentration. Which kit did you use?
2) Is your input DNA high molecular weight? It is absolutely crucial to have clean DNA with a fragment length of 100 kb plus ! Otherwise the tagmentation will give you very short DNA fragments which might not bind to the column in the clean up afterwards. As the incubation time of the tagmentation reaction influences the DNA fragment length you could play with the incubation time. But I would check the quality of the imput DNA first.
Let me know how it goes
Best Jan
Dear Jan
The purification was done with the alternative protocol using the AMPure XP Beads because the Zymo kit they don´t have here, but if you say it´s strongly recommended to use that kit, I will see if they can buy it.
The DNA to sequence is a bacterial genome, Rhodococcus genus, so I guess we are on the safe side with the size of the input DNA.
We wil try again, if we can buy the Zymo kit better, and I will let you know about the outcome.
I really appreciate your help as well as Shigehiro´s help. Hopefully you can give us some recommendations during our next trial. I´ll keep you posted.
Regards,
Susana
Dear Susana,
I would check the imput DNA on a gel to confirm the quality. You would expect a big band high up and no smear. It is not much work and you are on the safe side before starting the next trial.
Cheers Jan
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