Tweet
Hi,
I would like to ask for some help troubleshooting library preparation from bacterial genomes.
We are using Illumina's Nextera XT kit, strictly following the kit's protocol, and even using the same consumables as suggested there. DNA was extracted using Epicenter gram positive kit and checked using Nanodrop and Qubit. Before proceeding to Miseq run, we checked our libraries after PCR clean up (AMPure beads) using Tapestation with a HS D1000 ScreenTape. After a first quite unsuccessful trial, we ran the protocol again from the beginning, but using the same DNA samples. Attached are a figure of the Tapestation gel (1st run above, 2nd run below, samples in same order), and a table with the DNA sample parameters. I could send the histogram if needed (I don't know how to embed the figures in the message).
From those samples with "some" library, what samples would be OK for sequencing in your opinion?
And what could be going wrong with the other samples? Too little DNA? No tagmentation, no PCR or clean up problems?
Any help and tips would be greatly appreciated :-)
I would like to ask for some help troubleshooting library preparation from bacterial genomes.
We are using Illumina's Nextera XT kit, strictly following the kit's protocol, and even using the same consumables as suggested there. DNA was extracted using Epicenter gram positive kit and checked using Nanodrop and Qubit. Before proceeding to Miseq run, we checked our libraries after PCR clean up (AMPure beads) using Tapestation with a HS D1000 ScreenTape. After a first quite unsuccessful trial, we ran the protocol again from the beginning, but using the same DNA samples. Attached are a figure of the Tapestation gel (1st run above, 2nd run below, samples in same order), and a table with the DNA sample parameters. I could send the histogram if needed (I don't know how to embed the figures in the message).
From those samples with "some" library, what samples would be OK for sequencing in your opinion?
And what could be going wrong with the other samples? Too little DNA? No tagmentation, no PCR or clean up problems?
Any help and tips would be greatly appreciated :-)