libraries preparation from LCM-FFPE
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Erin Wu
Mar 19, 2021, 1:30:21 AM3/19/21
to Smart-3SEQ
Hi, Joe.
I microdissected about 400 cells from formalin-fixed paraffin embedded tissue, then constructed a single library using Smart-3SEQ methods FFPE version, and the PCR cycle is 22. But the size of distribution is seemingly strange as shown below. There are three main peaks at 219, 285, 502. I have no idea what's the reason. I repeated the experience carefully and tried to reduce the cycles adoubting much more PCR cycles , but still get the same result. Can you share your experience about that? Thank you so much!
That would be good if you can provide your size of ditribution from LCM-FFPE sample libraies.
Joe Foley
Mar 19, 2021, 1:03:53 PM3/19/21
to smart...@googlegroups.com
This library appears severely overamplified. The wide peaks around
500 and 10,000 bp are almost certainly overamplification artifacts
rather than long cDNA inserts, and it's possible the peak at 285 is
an artifact as well. With this much overamplification it's hard to
tell how much of your yield is good library inserts vs. adapter
dimers, but at least it's a good sign there was enough successful
library material to overamplify.
If these aren't precious samples, I would recommend a calibration experiment: dissect several samples of about 400 cells and do the library preps side-by-side with different numbers of PCR cycles. According to our formula calibrated on human RNA you should only need 17 cycles (assuming 10 pg per cell), though usually you need one or two extra cycles with FFPE tissue and it depends on the cell type so it's preferable to calibrate it yourself.
Or if you already have a lot of libraries like this and they're too precious to redo, overamplification doesn't necessarily imply they'll fail in sequencing. You can't quantify libraries with a profile this messy using the Bioanalyzer (too many artifacts), SYBR qPCR (depends on the Bioanalyzer for length estimation), or even Qubit (single-stranded artifacts won't bind the fluorescent label) so you'd have to use TaqMan qPCR to figure out how much to load. If your circumstances allow it, you could do a cheap test run with a MiSeq Nano kit or spike the libraries into another pool that doesn't have conflicting indexes to get some sequencing QC metrics without committing to an expensive run.
If these aren't precious samples, I would recommend a calibration experiment: dissect several samples of about 400 cells and do the library preps side-by-side with different numbers of PCR cycles. According to our formula calibrated on human RNA you should only need 17 cycles (assuming 10 pg per cell), though usually you need one or two extra cycles with FFPE tissue and it depends on the cell type so it's preferable to calibrate it yourself.
Or if you already have a lot of libraries like this and they're too precious to redo, overamplification doesn't necessarily imply they'll fail in sequencing. You can't quantify libraries with a profile this messy using the Bioanalyzer (too many artifacts), SYBR qPCR (depends on the Bioanalyzer for length estimation), or even Qubit (single-stranded artifacts won't bind the fluorescent label) so you'd have to use TaqMan qPCR to figure out how much to load. If your circumstances allow it, you could do a cheap test run with a MiSeq Nano kit or spike the libraries into another pool that doesn't have conflicting indexes to get some sequencing QC metrics without committing to an expensive run.
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