Post-PCR: Prepare for Sequencing

“Sub-Pools”

After completing all PCRs, gel electrophoresis and gel densitometry measurements, we can then pool isolates together at equimolar concentrations of 200 ng/μL. Here we wish to combine all isolates with unique MIDs per marker into one pool.

⚠️ REMEMBER: Ensure that there is only ONE of each MID per pool: this is important when demultiplexing bioinformatically.

Once you have collated all of your absolute quantity data, you can then generate a table as below. Note, each pool should have AT LEAST ONE POSITIVE CONTROL, e.g., 3D7. Each isolate genotyped with K13-1 with a unique MID are added to a unique “sub-pool”, in this case labelled “1A”.

Table 10. Example table to organise pooling of isolates per marker in 'sub-pools'
Sample Name	MID	Marker	Pool	Plate Position	DNA concentration^a	Volume (μL) to add to pool	Cumulative volume
Isolate 1	1	K13-1	1A	2A	62.65	3.2	3.2
Isolate 2	2	K13-1	1A	2B	65.74	3.0	6.2
Isolate 3	3	K13-1	1A	2C	129.47	1.5	7.7
Isolate 4	4	K13-1	1A	2D	92.17	2.2	9.9
Isolate 5	5	K13-1	1A	2E	144.59	1.4	11.3
3D7	108	K13-1	1A	7A	129.00	1.6	12.9
^a Agarose gel densitometry data

Next, you can calculate the total volume (μL) (12.9) and total DNA concentration (ng/μL) (623.62). This information is relevant for the following section.

“Master-Pools”

Now that we have our “sub-pools”, we can add all of our “Pool 1’s” for each marker together. However, before proceeding, we need to offset for fragment length to ensure that one marker isn’t preferentially sequenced over another. For the ALARMcoding protocol, this is our offsetting calculation:

Table 11. Offsetting by marker fragment size.
Marker	Size (bp)	Size + MID (bp)	Offset
mdr1-3	552	562	1.000
dhfr	523	533	0.948
dhps-2	521	531	0.945
dhps-1	493	503	0.895
mdr1-2	488	498	0.886
aat1	448	458	0.815
pfs47-2	429	439	0.781
mdr1-1	413	423	0.753
K13-1	402	412	0.733
pfs47-1	390	400	0.712
K13-2	323	333	0.593
crt	214	224	0.399

Armoured with this, we can create our “master-pools” containing one “sub-pool” of each marker.

Table 12. Example table to organise pooling of isolates per marker in 'sub-pools'
Pool	Sub-Pool	Isolates	Marker	Amplicon Size (bp)	Pool Offset Ratio	Sub-pool Total Volume (μL)	DNA per sub-pool (ng)	Sub-pool Total DNA (ng/μL)	DNA per sub-pool required for master-pool (ng)	Volume per sub-pool required for master-pool (ng)
1	1E	104	mdr1-3	562	1.0000000	296.3	20800	70.19912	5000.000	71.22596
1	1G	104	dhfr	533	0.9483986	322.4	20800	64.51613	4741.993	73.50089
1	1I	104	dhps-2	531	0.9448399	323.2	20800	64.35644	4724.199	73.40679
1	1H	104	dhps-1	503	0.8950178	299.6	20800	69.42590	4475.089	64.45849
1	1D	104	mdr1-2	498	0.8861210	449.9	20800	46.23250	4430.605	95.83313
1	1L	104	aat1	458	0.8149466	460.1	20800	45.20756	4074.733	90.13388
1	1K	104	pfs47-2	439	0.7811388	410.9	20800	50.62059	3905.694	77.15623
1	1C	104	mdr1-1	423	0.7526690	285.0	20800	72.98246	3763.345	51.56507
1	1A	104	K13-1	412	0.7330961	266.1	20800	78.16610	3665.480	46.89348
1	1J	104	pfs47-1	400	0.7117438	356.7	20800	58.31231	3558.719	61.02861
1	1B	104	K13-2	333	0.5925267	613.9	20800	33.88174	2962.633	87.44042
1	1F	104	crt	224	0.3985765	474.8	20800	43.80792	1992.883	45.49138
^a Calculated as the number of isolates per sub-pool (104) times the 200 ng/uL per amplicon equimolar concentration standardisation: 20,800 ng total

Now add these volumes together to create the master-pool!

Purification and Quantification

These “master-pools” were then purified using a Agencourt AMPure XP magnetic beads at 1.8X bead-to-sample ratio as described by the manufacturer (Beckman Coulter) and quantified using a PicoGreen Assay as described by the manufacturer (Thermo Scientific).

Now you’re ready to send it off for sequencing!