hyunminkang
commented
3 years ago Have you compared the VCFs generated from freemuxlet with the original genotypes? Would it be possible that the data is a lot of ambient mRNAs so that ambient RNAs were represented as one cluster? freemuxlet tried to avoid such a case, but it may be imperfect.
Thanks, Hyun.
Hyun Min Kang, Ph.D. Associate Professor of Biostatistics University of Michigan, Ann Arbor Email : hmkang@umich.edu
On Mon, Feb 15, 2021 at 5:23 PM xmignot notifications@github.com wrote:
Hi, I'm trying to demultiplex the sequence results of a series of 10x experiments (both 3 and 5 chemistry). I started by using demuxlet (we have gwas data available for the samples), but also ran freemuxlet using 1000 genomes VCF filtered as described in the tutorial as a reference. We additionally have multiseq results (a more involved demultiplexing protocol that I'm treating as ground truth) on just the 3 data. I'm a little concerned about the results from freemuxlet, as they appear to map very noisily to the demuxlet/multiseq sample ids. I built a mapping of consensus SNG barcodes between each protocol, and while demuxlet maps very cleanly to the multiseq labels in the 3 data for both the 3 and the 5 data the freemuxlet clusters are distributed across lots of sample ids. As an example, here are some rows from each mapping:
[demuxlet to multiseq] 109D12: ['109D12: 0.9238', '61C07: 0.0092', '119A02: 0.0074', '119A04: 0.0067', '113E02: 0.006'] ... [freemuxlet to multiseq] 6: ['61C04: 0.2754', '119A03: 0.2748', '119A02: 0.1642', '61D08: 0.1314', '119B12: 0.0609']
Do you have any advice on how to debug this or insights into what could be going on? I haven't tried passing the variant gwas positions used in demuxlet to freemuxlet as a reference, but I imagine this should give more consistent results. However, I want to be able to use the 1000 genomes variants as it seems this would be another way to independently validate the demultiplexed barcodes - additionally I've been advised they are probably more effective for freemuxlet. Thanks!
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xmignot
Hi, I'm trying to demultiplex the sequence results of a series of 10x experiments (both 3' and 5' chemistry). I started by using demuxlet (we have gwas data available for the samples), but also ran freemuxlet using 1000 genomes VCF filtered as described in the tutorial as a reference. We additionally have multiseq results (a more involved demultiplexing protocol that I'm treating as ground truth) on just the 3' data. I'm a little concerned about the results from freemuxlet, as they appear to map very noisily to the demuxlet/multiseq sample ids.
I built a mapping of consensus SNG barcodes between each protocol, and while demuxlet maps very cleanly to the multiseq labels in the 3' data for both the 3' and the 5' data the freemuxlet clusters are distributed across lots of sample ids.
As an example, here are some rows from each mapping:
Do you have any advice on how to debug this or insights into what could be going on? I haven't tried passing the variant gwas positions used in demuxlet to freemuxlet as a reference, but I imagine this should give more consistent results. However, I want to be able to use the 1000 genomes variants as it seems this would be another way to independently validate the demultiplexed barcodes - additionally I've been advised they are probably more effective for freemuxlet.
Thanks!