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 (ng)
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.

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

Going forward

1. Create covariate matrix and complete pairwise DML assessment in methylKit
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