Hi guys, I need quick help!
I'm sequencing with an Illumina Miseq for the first time. I'm following the Nextera XT workflow. My samples are S. aureus genomic DNA. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. I couldn't see any bands with my AMPure products by 2% AGE or Bioanalyzer, although Quantifluor/Qubit shows readings (ranging from 0.3-5 ng/uL). Is it possibly due to the detection limit of the machine (considering how low the starting amount is needed with the Nextera XT workflow), or do you think there is something wrong with my samples?
My initial genomic DNA samples were way high up than the recommended 0.2 ng/ul starting material so I had to dilute it down to get it around that ballpark range. I used Quantifluor for the quantification. It's possible I have thawed the original genomic DNA at least thrice in the dilution process. In the tagmentation step I loaded 5 - 10 uL of the diluted gDNA, depending on how lower than the target 0.2 ng/uL my sample was. I did the PCR clean-up following the 0.6x vol. recommended for >250bp. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. Since the product post-normalization is noted to be undetectable by Qubit or Bioanalyzer, I tried to QC my AMPure products instead. I tried viewing my PCR clean-up products by 2% AGE but did not see any bands. I quantified them by Quantifluor and got readings ranging from 1 to 6 ng/uL (Qubit gave 20-40% lower readings for select samples that I re-quantified; it's possible I started out with higher amounts than recommended). I ran 12 selected samples by Bioanalyzer and couldn't see a peak, maybe a very subtle one for two samples, which incidentally had the highest concentrations in the lot (2-3 ng/uL by Qubit, or 4-5 ng/uL by Quantifluor), around the 500 bp range. I don't know if the lack of peaks in the Bioanalyzer is due to a limitation in the sensitivity of the machine or if my samples are bad. Should I proceed or re-do the sample prep?
EDIT: My Nextera XT Sample Prep kit is expired by 3 months, but that has not been a problem in previous runs. My Bioanalyzer kit, too, by 1 year, if that could be a factor. I loaded only 1 uL of my sample into the Bioanalyzer chip (the concentration range was 0.3 to 3 ng/uL). All the undetectable samples had concentrations below 1 ng/uL by Qubit (1.6 ng/uL by QF).
I'm sequencing with an Illumina Miseq for the first time. I'm following the Nextera XT workflow. My samples are S. aureus genomic DNA. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. I couldn't see any bands with my AMPure products by 2% AGE or Bioanalyzer, although Quantifluor/Qubit shows readings (ranging from 0.3-5 ng/uL). Is it possibly due to the detection limit of the machine (considering how low the starting amount is needed with the Nextera XT workflow), or do you think there is something wrong with my samples?
My initial genomic DNA samples were way high up than the recommended 0.2 ng/ul starting material so I had to dilute it down to get it around that ballpark range. I used Quantifluor for the quantification. It's possible I have thawed the original genomic DNA at least thrice in the dilution process. In the tagmentation step I loaded 5 - 10 uL of the diluted gDNA, depending on how lower than the target 0.2 ng/uL my sample was. I did the PCR clean-up following the 0.6x vol. recommended for >250bp. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. Since the product post-normalization is noted to be undetectable by Qubit or Bioanalyzer, I tried to QC my AMPure products instead. I tried viewing my PCR clean-up products by 2% AGE but did not see any bands. I quantified them by Quantifluor and got readings ranging from 1 to 6 ng/uL (Qubit gave 20-40% lower readings for select samples that I re-quantified; it's possible I started out with higher amounts than recommended). I ran 12 selected samples by Bioanalyzer and couldn't see a peak, maybe a very subtle one for two samples, which incidentally had the highest concentrations in the lot (2-3 ng/uL by Qubit, or 4-5 ng/uL by Quantifluor), around the 500 bp range. I don't know if the lack of peaks in the Bioanalyzer is due to a limitation in the sensitivity of the machine or if my samples are bad. Should I proceed or re-do the sample prep?
EDIT: My Nextera XT Sample Prep kit is expired by 3 months, but that has not been a problem in previous runs. My Bioanalyzer kit, too, by 1 year, if that could be a factor. I loaded only 1 uL of my sample into the Bioanalyzer chip (the concentration range was 0.3 to 3 ng/uL). All the undetectable samples had concentrations below 1 ng/uL by Qubit (1.6 ng/uL by QF).
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