16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

Fouhy, Fiona; Clooney, Adam G.; Stanton, Catherine; Claesson, Marcus J.; Cotter, Paul D.

doi:10.1186/s12866-016-0738-z

Table 1 Details on number of sequencing reads, read lengths, percentage of reads retained post quality analysis

From: 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

Primer set	Raw	Quality	Length	Remaining	% Retained	After Chimera Removal	% Chimeras	% Retained
MiSeq
V4-V5
Mock DNA	47966	Q25	365–385	42701	89.023475	47966	0	100
Qiagen PBS^a
Qiagen glycerol	14071	Q25	365–385	13724	97.533935	13717	0.05100554	99.94899446
RBB PBS	18072	Q25	365–385	17253	95.4681275	18026	0.25453741	99.74546259
RBB glycerol	22650	Q25	365–385	20534	90.6578366	22626	0.10596026	99.89403974
V1-V2
Mock DNA	576244	Q25	305–325	310254	53.840734	308989	0.40773044	99.59226956
Qiagen PBS	206140	Q25	305–325	165035	80.05966	164117	0.55624564	99.44375436
Qiagen glycerol	274886	Q25	305–325	153566	55.86534	152034	0.99761666	99.00238334
RBB PBS	420677	Q25	305–325	327953	77.958386	324319	1.10808561	98.89191439
RBB glycerol	342405	Q25	305–325	189474	55.336224	181819	4.04013216	95.95986784
V1-V2 deg
Mock DNA	339219	Q25	305–325	164586	48.5190983	161382	1.94670264	98.05329736
Qiagen PBS	432220	Q25	305–325	170830	39.5238536	166923	2.28706902	97.71293098
Qiagen glycerol	277087	Q25	305–325	100478	36.262257	100031	0.4448735	99.5551265
RBB PBS	407061	Q25	305–325	111020	27.2735536	110057	0.86741128	99.13258872
RBB glycerol	373903	Q25	305–325	117567	31.4431818	116400	0.99262548	99.00737452
Ion PGM
V4-V5
Mock DNA	123511	Q25	420–440	57467	46.5278396	51923	9.64727583	90.35272417
Qiagen PBS	173203	Q25	420–440	74942	43.2683037	60366	19.4497078	80.55029223
Qiagen glycerol	194132	Q25	420–440	58267	30.0141141	49474	15.0908748	84.90912523
RBB PBS	211696	Q25	420–440	77227	36.4801413	68006	11.9401246	88.05987543
RBB glycerol	203949	Q25	420–440	84407	41.386327	71316	15.5093772	84.49062282
V1-V2
Mock DNA	389410	Q25	360–380	191184	49.0958116	190016	0.61092978	99.38907022
Qiagen PBS	21501	Q25	360–380	14852	69.0758569	14804	0.3231888	99.6768112
Qiagen glycerol	35900	Q25	360–380	26518	73.8662953	26418	0.37710235	99.62289765
RBB PBS	35343	Q25	360–380	19157	54.2030954	19046	0.57942267	99.42057733
RBB glycerol	62195	Q25	360–380	42150	67.7707211	42003	0.34875445	99.65124555
V1-V2 deg
Mock DNA	207570	Q25	360–380	75999	36.6136725	71500	5.91981473	94.08018527
Qiagen PBS	398459	Q25	360–380	236444	59.3396058	231427	2.12185549	97.87814451
Qiagen glycerol	439533	Q25	360–380	214562	48.8159023	208065	3.02802919	96.97197081
RBB PBS	376207	Q25	360–380	180289	47.9228191	174723	3.08726545	96.91273455
RBB glycerol	389283	Q25	360–380	184442	47.3799267	166537	9.70765878	90.29234122

^aDNA failed to amplify with V4-V5 MiSeq primers for the Qiagen PBS extracted cells so no sequencing data for this extraction sample

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ISSN: 1471-2180

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