KBay Bleaching Pairs 16S Processing

Processing with Ariana and Kevin’s samples. Spreadsheet for sample names and plate maps found here.

Putnam Lab 16S protocol found here.

Trial run

Master mix calculations:

	(ul)	# of rxns	(ul) to add
Phusion MM	12.5	43.5	543.75
806RB primer	0.5	43.5	21.75
515F primer	0.5	43.5	21.75
Ultra pure H2O	10.5	43.5	456.75

Platemap (plate 1)

	1	2	3	4	5	6	7	8	9	10	11	12
A	KW-10	KW-10	KW-10	KW-11	KW-11	KW-11	KW-12	KW-12	KW-12	KW-13	KW-13	KW-13
B	AH-2	AH-2	AH-2	AH-3	AH-3	AH-3	AH-4	AH-4	AH-4	AH-5	AH-5	AH-5
C	ES-2	ES-2	ES-2	ES-4	ES-4	ES-4	ES-6	ES-6	ES-6	ES-16	ES-16	ES-16
D	POS(+)	POS(+)	POS(+)	NEG(-)	NEG(-)	NEG(-)

Positive control: M. capitata sample from Holobiont Integration M2410 from well B1 from dilution plate 2.
Negative control: Ultrapure water

Initially we were worried about the difference in band size between KW and AH/ES samples (KW appears to be closer to 400 bp and AH/ES bands seem to be closer to 300 bp long). This could be because AH and ES samples are picking up more of the 18S and mitochondria regions than the 16S regions. However, the positive control was a sample that we have sequenced previously and in the gel image above it looks like the M. capitata ES samples.

Positive control M2410 sequencing details

M2410 = HPW060 (Sequencing ID with URI GSC)

Denoising stats: M2410 was the 57th lowest # of reads after filtering out non-chimeric out of 262 samples.

Plug ID	Sequencing ID	Input	Filtered	% of input passed filtered	Denoised	Merged	% of input merged	Non-chimeric	% of input non-chimeric
M2410	HPW060	31027	22804	73.5	22472	20306	65.45	19673	63.41
Average of all samples		40776.97	29593.68	72.49211	29411.86	27954.59	67.92323	27939.43	67.88873

Next steps could be to try a short run with more of the samples from Holobiont Integration - because I only did one of my pervious ones, I don’t know if the faint band is truly reflective of the below average denoising statistics above. We decided to move forward with the protocol as is.

Bacteria / Total:

• M2410: 64.11% of reads are bacteria (12,612 out of 19,673 reads)
• Average sample: 56.12% reads are bacteria

Chloroplast / Total:

• M2410: 340 reads are chloroplast (1.73%)
• Average sample: 2.62% reads are chloroplast

Plate 2, 3

Plate 2 (20211111)

	1	2	3	4	5	6	7	8	9	10	11	12
A	KW-10	KW-10	KW-10	23	23	23	35	35	35	46	46	46
B	11	11	11	24	24	24	36	36	36	49	49	49
C	12	12	12	29	29	29	37	37	37	51	51	51
D	13	13	13	30	30	30	39	39	39	53	53	53
E	14	14	14	31	31	31	40	40	40	54	54	54
F	15	15	15	32	32	32	41	41	41	55	55	55
G	21	21	21	33	33	33	43	43	43	56	56	56
H	22	22	22	34	34	34	44	44	44	57	57	57

Plate 3 (20211111)

	1	2	3	4	5	6	7	8	9	10	11	12
A	58	58	58	74	74	74	5	5	5	13	13	13
B	62	62	62	80	80	80	6	6	6	14	14	14
C	63	63	63	87	87	87	7	7	7	16	16	16
D	65	65	65	88	88	88	8	8	8	15	15	15
E	67	67	67	89	89	89	9	9	9	17	17	17
F	68	68	68	AH-2	AH-2	AH-2	10	10	10	18	18	18
G	71	71	71	3	3	3	11	11	11	19	19	19
H	73	73	73	4	4	4	12	12	12	20	20	20

Strip tubes for positive and negative control

POS(+) 1	POS(+) 1	POS(+) 1	POS(+) 2	POS(+) 2	POS(+) 2	POS(+) 3	POS(+) 3
POS(+) 3	NEG(-)	NEG(-)	NEG(-)

Master mix calculations:

	(ul)	# of rxns	(ul) to add
Phusion MM	12.5	215	2687.5
806RB primer	0.5	215	107.5
515F primer	0.5	215	107.5
Ultra pure H2O	10.5	215	2257.5

Positive control details:

POS(+) 1	M1095	Dilution plate 2 well D1
POS(+) 2	M1694	Dilution plate 2 well E1
POS(+) 3	M2986	Dilution plate 2 well F1

Plates were left at 4C instead of -20C over the weekend. The gel looks OK, I won’t redo these for now.

Plate 4, 5

Plate 4 (20211117)

	1	2	3	4	5	6	7	8	9	10	11	12
A	21	21	21	29	29	29	37	37	37	17	17	17
B	22	22	22	30	30	30	38	38	38	18	18	18
C	23	23	23	31	31	31	39	39	39	21	21	21
D	24	24	24	32	32	32	40	40	40	22	22	22
E	25	25	25	33	33	33	ES-2	ES-2	ES-2	23	23	23
F	26	26	26	34	34	34	4	4	4	24	24	24
G	27	27	27	35	35	35	6	6	6	25	25	25
H	28	28	28	36	36	36	16	16	16	26	26	26

Plate 5 (20211117)

	1	2	3	4	5	6	7	8	9	10	11	12
A	28	28	28	37	37	37	45	45	45	55	55	55
B	29	29	29	38	38	38	46	46	46	56	56	56
C	30	30	30	39	39	39	47	47	47	57	57	57
D	31	31	31	40	40	40	50	50	50	59	59	59
E	32	32	32	41	41	41	51	51	51	NEG(-)	NEG(-)	NEG(-)
F	33	33	33	42	42	42	52	52	52
G	34	34	34	43	43	43	53	53	53
H	35	35	35	44	44	44	54	54	54

No positive controls because these were in the first 2 plates already.

Master mix calculations:

	(ul)	# of rxns	(ul) to add
Phusion MM	12.5	193	2412.5
806RB primer	0.5	193	96.5
515F primer	0.5	193	96.5
Ultra pure H2O	10.5	193	2026.5

Plates left at 4C overnight and gel’d the next day.

Re-did the gel for some samples from plate 2. These look OK. Ready to submit all of the above samples.

Submit to Janet

Platemaps dropped off to Janet spreadsheet link.

We choose the code WSH (Wong Strand Huffmyer)001 - 096 for the first plate, 101-196 for the second plate, and 201-296 for the third plate.