Hawaii Gigas Methylation Analysis Part 10

WGS resequencing quote

I plan on extracting SNP data from my WGBS data, but Steven suggested I get a quote for WGS resequencing. This would give us more robust genotype data that we could use in our methylation analysis. While there is the possibility Sam or I could extract additional DNA, I first asked Sam if there was any leftover ctenidia DNA. He said there was and shared this lab notebook post with the yield for each sample. According to this other post, he used 1500 µg of DNA for each sample. I quickly calculated how much DNA is left for each sample.

Table 1. DNA left for each ctenidia sample

Sample_ID Concentration(ng/uL) Volume(uL) Total_DNA (ng) Amount Left
2N_HI_5 40.4 100 4040 2540
2N_HI_8 11.6 100 1160 0
2N_HI_9 32.3 100 3230 1730
2N_HI_10 61 100 6100 4600
2N_HI_11 21 100 2100 600
2N_HI_12 11.2 100 1120 0
2N_LOW_1 32.1 100 3210 1710
2N_LOW_2 32.5 100 3250 1750
2N_LOW_3 36 100 3600 2100
2N_LOW_4 40.2 100 4020 2520
2N_LOW_5 17.8 100 1780 280
2N_LOW_6 22.8 100 2280 780
3N_HI_2 29.6 100 2960 1460
3N_HI_3 71.8 100 7180 5680
3N_HI_5 29.3 100 2930 1430
3N_HI_8 38.9 100 3890 2390
3N_HI_10 38.3 100 3830 2330
3N_HI_11 52.3 100 5230 3730
3N_LOW_6 35.3 100 3530 2030
3N_LOW_7 43.6 100 4360 2860
3N_LOW_8 63.9 100 6390 4890
3N_LOW_10 54.4 100 5440 3940
3N_LOW_11 50.8 100 5080 3580
3N_LOW_12 52 100 5200 3700

Most samples have > 1000 µg of DNA, but there are three (2N_HI_11, 2N_LOW_5, 2N_LOW_6) that have less. Two samples (2N_HI_8 and 2N_HI_12) do not have any DNA left over, and they are both from the same treatment, so that could pose an issue. It’s interesting that the samples with less DNA are all diploid!

In any case, I submitted quotes to GENEWIZ and Northwest Genomics Center (NWGC). I asked how much DNA is required to for library preparation and sequencing. I spoke with Katie about WGS, and she suggested I base my quote on information from the Illumina coverage calculator. I’ll require need ~22 billion bases of output for the NovaSeq. I don’t know if GENEWIZ or NWGC use NovaSeq, but Katie thinks that sequencing will cost ~$6,000 for all samples.

Screen Shot 2021-03-05 at 12 24 09 PM

I’ll update this issue with information when I get it.

Going forward

  1. Try BS-SNPer and EpiDiverse for SNP extraction from WGBS data
  2. Investigate comparison mechanisms for samples with different ploidy in oysters and other taxa
  3. Test-run DSS and ramwas
  4. Transfer scripts used to a nextflow workflow
  5. Update methods
  6. Update results

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Hawaii Gigas Methylation Analysis Part 9

Preliminary DML identification

Diploid vs. triploid

Low pH vs. ambient pH

Going forward

  1. Get WGS resequencing quote
  2. Try BS-SNPer and EpiDiverse for SNP extraction from WGBS data
  3. Investigate comparison mechanisms for samples with different ploidy in oysters and other taxa
  4. Test-run DSS and ramwas
  5. Transfer scripts used to a nextflow workflow
  6. Update methods
  7. Update results

Please enable JavaScript to view the comments powered by Disqus.

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via IFTTT