Determine the optimum w and k sizes

[mbhall88]

Do a sweep through pandora window and kmer sizes and see which result in the best drprg performance for Illumina and Nanopore

[mbhall88]

The way this analysis was setup is I took 183 isolates from the head to head dataset which have a minimum of 3x coverage for both Illumina and Nanopore.

I then run drprg on those isolates with the following window and kmer sizes (no combinations where w>k though)

w

- 10  # default (nanopore) in minimap2
- 11  # short read default in minimap2
- 14  # current default
- 19  # best illumina found in Rachel's thesis

k

- 15  # current default
- 17
- 19
- 21  # value used for short reads by minimap2
- 25
- 31  # best illumina value found in Rachel's thesis

I then aggregate the number of false positive and negative resistance calls across all drugs and plot them.

In the above plot, the dashed lines with the triangle points are FP counts and the solid lines with circle points are FN counts. The different colours indicate the window size, with kmer size on the x-axis.

Seems like something weird has happened in both technologies at k-17 w=14 as the FPs randomly sky-rocket. I will look into what has caused this because based on the trends, this looks like it might have been the best combination for both technologies. As it stands k=17 and w=11 looks to be the best.

[mbhall88]

The jump in FNs are effectively all in Rifampicin and specifically in the mutation rpoB_S450X. What is happening (in both technolopgies) is that for some reason at that precise (w, k) pairing, we start getting hits on the reference allele, which leads to the FRS (fraction of read support) filter being applied. There are no hits on the reference allele for k=15 and k=19 at that w=14 - for the isolates I looked at.

Example VCF entires (I have removed excess VARID and PREDICT values)

sample mada_1-5 nanopore w=14 k=15

rpoB    1449    28f3a195        C       T       .       PASS    SVTYPE=SNP;GRAPHTYPE=SIMPLE;VARID=rpoB_S450X;PREDICT=R        GT:MEAN_FWD_COVG:MEAN_REV_COVG:MED_FWD_COVG:MED_REV_COVG:SUM_FWD_COVG:SUM_REV_COVG:GAPS:LIKELIHOOD:GT_CONF    1:0,25:0,27:0,35:0,33:0,77:0,81:1,0.333333:-421.469,-43.1295:378.339

sample mada_1-5 nanopore w=14 k=17

rpoB    1449    f431d8b5        C       T       .       frs     SVTYPE=SNP;GRAPHTYPE=SIMPLE;VARID=rpoB_S450X;PREDICT=R        GT:MEAN_FWD_COVG:MEAN_REV_COVG:MED_FWD_COVG:MED_REV_COVG:SUM_FWD_COVG:SUM_REV_COVG:GAPS:LIKELIHOOD:GT_CONF    1:18,38:18,42:18,38:18,42:37,77:37,84:0.5,0:-434.949,-169.272:265.677

sample mada_1-5 nanopore w=14 k=19

rpoB    1449    a0e01ee5        C       T       .       PASS    SVTYPE=SNP;GRAPHTYPE=SIMPLE;VARID=rpoB_S450X;PREDICT=R        GT:MEAN_FWD_COVG:MEAN_REV_COVG:MED_FWD_COVG:MED_REV_COVG:SUM_FWD_COVG:SUM_REV_COVG:GAPS:LIKELIHOOD:GT_CONF    1:0,38:0,42:0,38:0,42:0,77:0,85:1,0:-538.414,-3.26102:535.153

For @lachlancoin's understanding, what you want to focus on here for the coverage is the MEAN_FWD_COVG and MEAN_REV_COVG fields

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I don't really get why this particular window size (14) at that particular kmer size (17) would cause this. But as this allele is inside the RRDR there could be something funky happening in the graph surrounding this position.

I'm going to add in w=13 and w=15 to get a better idea of exactly where this breaks down.

Any other ideas?

[mbhall88]

Waiting on resolution of iqbal-lab-org/pandora#293 as a few samples failed for that reason

[iqbal-lab]

How is the graph built? Are you starting with a list of variants/vcf and implicitly allowing loads of recombination, or starting with an MSA of known haplotypes and using make-prg? And does drprg do de novo via de bruin or racon, or neither?

[mbhall88]

How is the graph built? Are you starting with a list of variants/vcf and implicitly allowing loads of recombination, or starting with an MSA of known haplotypes and using make-prg?

I started with the cryptic VCF, subsampling 50 isolates from each lineage. Then apply the haplotype for each isolate to H37Rv. Then MSA that, then make_prg. I then run de novo (dBG) on that graph, update the graph with make prg, and then I hit the error when genotyping with pandora map. I only get it on 4 w-k combination, and, interestingly, all are when w=k. I don't hit this for any other w-k combination. In total, with the 181 isolates across the two sequencing modalities there are ~7-8k jobs in the pipeline and only 4 hit this error. I'll skip them for now, but it seems important to understand what is causing this.

[mbhall88]

I'm going to add in w=13 and w=15 to get a better idea of exactly where this breaks down.

Here is the plot with the extra window and kmer sizes.

tl;dr k=17 and w=11 seems best for both technologies

[mbhall88]

After the work to-date on #11 these results have changed quite noticeably - for FPs mainly.

After ranking the F1 scores for all of these combinations (and eyeballing) it seems the original default of w=14 and k=15 gives the best results. I'll revert back to that.

[mbhall88]

After all the recent work I thought I would revisit this.

Looking at the raw values I thought I'd try out w=11 and k=15 (currently the default is w=14 k=15) and I get the following diff for Illumina (no change for nanopore)

Tool	Drug	ΔFN	ΔFP
drprg	Amikacin	-2	0
drprg	Capreomycin	-1	0
drprg	Delamanid	0	-1
drprg	Ethambutol	-1	2
drprg	Ethionamide	-3	-7
drprg	Isoniazid	0	0
drprg	Kanamycin	-2	0
drprg	Levofloxacin	1	2
drprg	Linezolid	0	0
drprg	Moxifloxacin	-1	-1
drprg	Ofloxacin	1	0
drprg	Pyrazinamide	0	-1
drprg	Rifampicin	-1	0
drprg	Streptomycin	-3	0

So I think I'll stick with w=11 and k=15