Hello,
I would like to ask for some advice on normalization of Nextera XT libraries that will be sequenced on Miseq (paired ends 2x150). We got low output (~400K) in one run with our samples (bacterial genomes, ~2.5 Mb), and we believe something may not have worked well during or after the normalization step. So this time we want to do the normalization without following Nextera XT's protocol, that is, by quantitation of libraries with Qubit after clean up and Tapestation check.
1. To what concentration should we bring our libraries?
2. In what buffer should we dilute them?
3. Should this be done before denaturation or after?
4. How long can libraries (after PCR clean up, before normalization) be stored frozen before normalization etc. (in case we want to use our previous libraries and only normalize etc. them now)?
5. If someone has a protocol I would be glad to take a look because Illumina has many pieces of protocols and sheets which are a bit confusing to follow...
Attached are the last libraries we tried to run. For some reason we do not get longer libraries than these. Concentration of most of them seem ok, although we only checked them with Tapestation and not with Qubit last time. We are aware that some libraries are of low concentration. Is it possible that the addition of these low concentration libraries to beads normalization dropped the overall concentration even of the good libraries?
Since we want to sequence a lot of genomes, I wish we could use beads normalization. In your experience, would that work if we did not add the very low concentration libraries or dilutes the high concentration ones before this step?
Attached are some graphs from the last run. The only thing we thought about was the normalization and on steps.
Thanks a lot in advance!
I would like to ask for some advice on normalization of Nextera XT libraries that will be sequenced on Miseq (paired ends 2x150). We got low output (~400K) in one run with our samples (bacterial genomes, ~2.5 Mb), and we believe something may not have worked well during or after the normalization step. So this time we want to do the normalization without following Nextera XT's protocol, that is, by quantitation of libraries with Qubit after clean up and Tapestation check.
1. To what concentration should we bring our libraries?
2. In what buffer should we dilute them?
3. Should this be done before denaturation or after?
4. How long can libraries (after PCR clean up, before normalization) be stored frozen before normalization etc. (in case we want to use our previous libraries and only normalize etc. them now)?
5. If someone has a protocol I would be glad to take a look because Illumina has many pieces of protocols and sheets which are a bit confusing to follow...
Attached are the last libraries we tried to run. For some reason we do not get longer libraries than these. Concentration of most of them seem ok, although we only checked them with Tapestation and not with Qubit last time. We are aware that some libraries are of low concentration. Is it possible that the addition of these low concentration libraries to beads normalization dropped the overall concentration even of the good libraries?
Since we want to sequence a lot of genomes, I wish we could use beads normalization. In your experience, would that work if we did not add the very low concentration libraries or dilutes the high concentration ones before this step?
Attached are some graphs from the last run. The only thing we thought about was the normalization and on steps.
Thanks a lot in advance!
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