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Dear all,
Anyone has such experience? How to conquer the issue of low input DNA?
Thanks,
Wei
Anyone has such experience? How to conquer the issue of low input DNA?
Thanks,
Wei
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Make sure to have Illumina data from the same DNA prep for efficient error-correction
Two points:
1. To get more DNA try making the sample prep more efficient/use more starting material/
2. To allow efficient error-correction of the pacbio reads also prep PCR-free (optional: + Nextera matepair) libraries from the same lot of DNA as used for creating the PAC-Bio libraries, and sequence them at 30X coverage compared to the volume of the raw pacbio data you are getting.
First do self-errorcorection of the pacbio subreads from the same template,
than error correct the filtered pacbio subreads using the Illumina data before the assembly.
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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