Hi!
First of all, if this belongs in the Sample Prep / Library Generation forum, feel free to move it there.
Now on to my questions. Until now, I was only part of the bioinformatics of sequencing. However, I recently switched institutes and now I have to establish sequencing in my new lab. We bought a MiSeq and would like the students to participate in this field, too, which is why we offer a practical course in sequencing.
Given the limited time of such a course (~4 x 6-8 h), I chose the Nextera XT kit and would like to sequence some bacterial genomes for demonstration and a bit of computational analysis. The genomes will be bought (are there any limitations?) or prepped in advance, so that we can immediately start with the tagmentation reaction.
Given the fact that the Nextera XT kit is optimized for plate usage, which is rather unsuitable for a practical course, I'd like to change everything to tubes, except for maybe the magnetic bead parts. I don't think this should affect the protocol (as long as we keep track of the indices). Or are these false conclusions?
Do you have any experience in using PCR purification kits other than the Illumina-recommended AMPure XP? E.g. standard centrifuge kits? This would be much more suitable for the course.
Furthermore, what do you guys think would be best for sequencing the libraries? I only have two sensible options (considering the time needed for sequencing). The first one is the v2 kit with 50 cycles and 1x36 or 2x25 and the second one is the v3 kit with 150 cycles and 2x75. I tend toward the v2 kit with 50 cycles because it takes less time than the v3 and guarantees to finish o/n.
I hope it's not too much text and appreciate every feedback
First of all, if this belongs in the Sample Prep / Library Generation forum, feel free to move it there.
Now on to my questions. Until now, I was only part of the bioinformatics of sequencing. However, I recently switched institutes and now I have to establish sequencing in my new lab. We bought a MiSeq and would like the students to participate in this field, too, which is why we offer a practical course in sequencing.
Given the limited time of such a course (~4 x 6-8 h), I chose the Nextera XT kit and would like to sequence some bacterial genomes for demonstration and a bit of computational analysis. The genomes will be bought (are there any limitations?) or prepped in advance, so that we can immediately start with the tagmentation reaction.
Given the fact that the Nextera XT kit is optimized for plate usage, which is rather unsuitable for a practical course, I'd like to change everything to tubes, except for maybe the magnetic bead parts. I don't think this should affect the protocol (as long as we keep track of the indices). Or are these false conclusions?
Do you have any experience in using PCR purification kits other than the Illumina-recommended AMPure XP? E.g. standard centrifuge kits? This would be much more suitable for the course.
Furthermore, what do you guys think would be best for sequencing the libraries? I only have two sensible options (considering the time needed for sequencing). The first one is the v2 kit with 50 cycles and 1x36 or 2x25 and the second one is the v3 kit with 150 cycles and 2x75. I tend toward the v2 kit with 50 cycles because it takes less time than the v3 and guarantees to finish o/n.
I hope it's not too much text and appreciate every feedback
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