DNA Requirements for 16S rRNA Gene Analysis

Exectuve Summary

This pilot study is a follow-up on the Comparison of DNA Extraction Methods

The ZymoBIOMICS Microbial Community Standard is comprised of 10 microbes in known quantities. The Community Standard was diluted from neat to 1:1000000 to determine at what point the extracted DNA was no longer detected by Nanodrop analysis. Using the approximate cell concentration of the ZymoBIOMICS Microbial Community Standard provided by Zymo Research, dilutions could be converted to cell number for application in ‘real world’ studies. DNA extracted using the optimum homogenisation method and preferred extraction kit, identified in DNA extraction methods pilot study, was used. At the dilutions 1:100 to 1:1000000, DNA concentrations were recorded as being low. 16S amplification and library preparation was performed on these samples using the YouSeq NGS 16S kit. Products of the 16S amplification ran on a gel showed a DNA band down to a dilution of 1:100000, indicating that very low input DNA could still be amplified and detected with 16S PCR. This library was then pooled and sequenced. Sequencing of the pooled library revealed that all of the microbes within all dilutions of the ZymoBIOMICS Microbial Community Standard could be detected at the genus level. Further, only a decrease in percentage of reads was observed at dilutions of 1:100000 and 1:1000000, corresponding to approximately 28000 and 2800 cells respectively. The PCR no template control showed no reads assigned to that sample, while the kit only control showed some low-level contamination, indicating some potential experimental contamination, either environmental or cross. Adjusting for the mean number of reads showed that the number of reads returned for the PBS kit control was negligible and that the ZymoBIOMICS microbial community standard can be diluted down to 280000 bacterial cells and provide adequate results by 16S sequencing.

Comparison of DNA Extraction Methods

Executive Summary

The pilot study reported here aimed to determine which commercially available DNA extraction kit and homogenisation step performed the best when extracting DNA from the ZymoBIOMICS Community Standard, which is comprised of 10 microbes in known quantities.

As the ZymoBIOMICS standard is a known defined community, the best extraction kit and homogenisation method will be the protocol which results in the sequencing data closest to the initial input. The Community Standard was diluted from neat to 1:1000000 to determine if a particular extraction kit or homogenisation step yielded greater DNA concentrations and at what point DNA was no longer detected from each variable. DNA quantification was performed on a Nanodrop and all kits showed recovery of good quantity and quality DNA from both kits and both homogenisation steps. For all conditions, DNA concentration hit a base level at a 1:100 dilution.

Shotgun sequencing was performed on one replicate of the undiluted and 1:10 dilution of each extraction method and homogenisation method. From this data, we observed all conditions tested recovered all of the microbes which make up the community standard. PCA analysis showed that sample concentration (undiluted vs 1:10) or extraction kit (Qiagen vs ZymoBIOMICS) showed no significant difference in microbial DNA recovered. A significant difference in the microbial DNA composition was observed between homogenisation protocols (Tissue Lyser II vs Vortex Adapter).

Detecting Contaminants in 16S Data

Executive Summary

It is common to sequence multiple projects together on a single run in order to obtain optimal numbers of sequenced reads per sample whilst minimising data redundancy. This report I explore and validate methods to mitigate any downstream effects cross-project contamination may have. The dataset used in this study included human stool (CMS001), two defined mouse microbiomes (GFU005, Nanopore), a DNA standard obtained from Zymo (DNA_Std), and a specific pathogen-free mouse microbiome (SPF).

In general, about 20-40 ASVs were identified as cross-project contaminants. However, the diversity of the samples are still overestimated, as evidenced by the DNA standards and the MM12 experiments, suggesting that this method may still be too conservative. A follow-up report will be conducted on removing contaminants introduced at the bench level (kit contaminants, PCR, library preparation, etc.)

Evidence of cross-project contamination

The DNA Standards and GFU005 project allow us to measure the extent of cross-project contamination, as the theoretical composition of those samples are known. Table 1 shows the number of ASVs observed in each project, as compared to the expected number of ASVs, and the number of ASVs identified in this report as project contaminants.

Summary of number of ASVs in each project.

expt	n_sample	n_expected	n_observed	n_contam
dna_std	2	10	161	18
Nanopore	4	12	121	35
SPF	8	NA	490	21
GFU005	112	12	1514	43
CMS001	116	NA	3804	22

Project prevalence

It is expected that cross-project contaminations would occur rarely, and randomly. Therefore, ASVs that are observed in a small percentage of samples within a project are good candidates for cross-project contaminants. The threshold for what is considered “low prevalence” was determined by tallying hte number of ASVs observed at 5% prevalence, 4% prevalence and so on. The point where a large drop off of ASV frequencing is used as the threshold prevalence level. For example, Table 2 shows a prevalence of 2% is can be considered as “low prevalence” in both CMS001 and GFU005

Number of ASVs observed at each prevalence level.

Sample Prevalence	CMS001	GFU005
0.01	1817	953
0.02	2285	1110
0.03	2580	1179
0.04	2910	1234
0.05	3018	1267

Identifying candidate contaminants based on prevalence does not work for projects with few samples, as is the case with the DNA standards (2 samples), SPF (8 samples), and “Nanopore” project (4 samples). In these cases, candidate contaminants can be identified using a read count cut-off of 0.1% of the total abundance in the project. For the DNA standards, the total abundance (of ASVs expected to be in the standard, based on genus) was 94320. Therefore, ASVs with less than on equal to 94 reads were considered as candidate contaminants for the DNA standards. Contaminants to the Nanopore and SPF projects were identified in a similar way, 0.1% of where total abundance in the project was used to set cutoff thresholds for contaminants at 212 and 398 for Nanopore and SPF, respectivley.

Applying these threshold cutoffs to their respective projects results in identifing candidate project contaminats (Table 3). Next these low abundance or low prevalence ASVs warrented further investigation into their likelihood of originating from another project from the same sequencing run.

Number of candidate contaminants based on low abundance cut-off or low prevalence cut-off.

experiment	num. ASVs	num. candidate contaminants	cutoff method
Nanopore	121	83	low abundance
dna_std	161	101	low abundance
SPF	490	119	low abundance
GFU005	1514	91	low prevalence
CMS001	3804	106	low prevalence

Cross-Project comparison of ASV abundance

It is expected ASVs originating from other projects (rather than the current project) would be very abundant in other projects and rare in the current project. Therefore, these cross-project contaminants would have a very high fold change. Furthermore, the occurance of cross-project contaminants should be rare. Therefore, ASVs with large fold-changes and low frequencies can be considered as cross-project contaminants. Fold-changes were calculated between CMS001 and the project with the highest abundnaces for a given ASV. Fold-changes were calculated as “Other Project” / “Current Project”, so a high fold-change indicates that the ASV was abundant in the other project, and minimally present in current project of interest.

Comparisons of the candidate contaminants across all projects can be summarized by looking at how common these fold changes are. The fold changes of candidate contaminants identified for each project are tallied, and the frequencies are summarized in Tables 5-9. Based on the frequency of fold changes, cut-off for what is considered as a ‘large-fold’ difference is evident. These cut-offs are summarized in Table 4.

Fold change threshold for identifying project contaminants.

expt	threshold
dna_std	80
Nanopore	40
SPF	60
GFU005	60
CMS001	30

Tally of fold changes of candidate contaminants to DNA Standards.

Fold change (bin upper bound)	Number of ASVs
10	14
20	23
30	15
40	15
50	6
60	5
70	4
80	1
90	1
100	1
110	1
180	1
200	1
220	1
230	1
270	1
330	1
370	1
460	1
570	1
740	1
900	1
950	1
1270	1
3910	1
5030	1

Tally of fold changes of candidate contaminants to “Nanopore” project.

Fold change (bin upper bound)	Number of ASVs
10	21
20	16
30	9
40	2
50	2
60	1
70	1
80	2
90	1
100	1
140	2
160	1
190	1
210	1
220	1
230	1
250	1
260	1
280	2
300	1
330	1
350	2
390	1
420	2
450	1
460	1
470	1
510	1
590	1
640	1
670	1
1620	1
11480	1

Tally of fold changes of candidate contaminants to SPF project.

Fold change (bin upper bound)	Number of ASVs
10	36
20	18
30	19
40	14
50	10
60	1
70	2
80	2
90	2
110	1
160	1
230	1
390	1
400	1
430	1
750	1
790	1
3330	1
4020	1
7220	1
21190	1
23790	1
34610	1
35020	1

Tally of fold changes of candidate contaminants to GFU005 project.

Fold change (bin upper bound)	Number of ASVs
10	25
20	8
30	7
40	3
50	4
60	1
70	1
80	1
90	2
100	1
180	1
210	1
220	1
300	1
340	1
430	1
530	1
670	1
1100	1
1110	1
1730	1
1950	1
2610	1
2650	1
2850	1
2920	1
3050	1
3060	1
3650	1
3690	1
3720	1
3800	1
4140	1
4520	1
5720	1
6150	1
6160	1
6860	1
7810	1
9360	1
10810	1
11300	1
11680	1
13660	1
18340	1
20360	1
22530	1
41070	1

Tally of fold changes of candidate contaminants to CMS001 project.

Fold change (bin upper bound)	Number of ASVs
10	59
20	19
30	6
40	3
50	2
60	2
70	1
80	1
90	4
130	1
150	1
190	1
240	1
250	1
740	1
800	1
1390	1
4030	1

Contaminants identified for DNA standards. Total abundance for each ASV within the projects are shown.

featureID	CMS001	dna_std	GFU005	Nanopore	SPF
ASV103	211	11	893	2	62
ASV141	17364	81	2303	0	163
ASV163	274	35	1031	0	3404
ASV167	4788	27	538	0	12
ASV194	62	13	240	0	7327
ASV199	27343	7	298	0	0
ASV201	16075	17	545	0	4
ASV203	103	9	378	0	911
ASV206	101	8	312	0	1575
ASV208	9	3	61	0	2198
ASV21	223	36	45474	2476	0
ASV211	8	5	94	0	4484
ASV218	6	4	36	0	1466
ASV241	320	30	612	0	9882
ASV242	3796	17	156	0	17
ASV243	31093	68	460	0	11037
ASV245	4468	17	375	0	1131
ASV70	321	16	1189	7	80352

The taxonomic classifications associated with the ASVs identified as likely cross-project contaminants for DNA standards.

featureID	Taxon
ASV103	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV141	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV163	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV167	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnospiracea incertae sedis;s__NA
ASV194	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV199	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Peptostreptococcaceae;g__Clostridium XI;s__NA
ASV201	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_distasonis(AB238922)
ASV203	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV206	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV208	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV21	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV211	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV218	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Anaerotruncus;s__NA
ASV241	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV242	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_distasonis(AB238922)
ASV243	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Clostridium XlVa;s__NA
ASV245	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Intestinimonas;s__NA
ASV70	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_apodemi(AJ871178)

</tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tbody> </table></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul>

ASVs remaining in DNA standards after removal of contaminants. Expected member of DNA standard are demarked with *. Total abundance for ASVs for each project shown.

featureID	expected_member	dna_std	Nanopore	SPF	GFU005	CMS001
ASV10	</td>	8889	395	184	8310	122
ASV101		62	6	0	796	291
ASV106		95	7	27	2276	870
ASV110		113	6	1570	4018	1594
ASV118		383	7	6831	11339	3949
ASV120		41	5	61	1500	513
ASV122	</td>	15195	0	0	4	11
ASV123	</td>	13128	0	0	12	0
ASV124	</td>	12401	0	0	3	0
ASV125	</td>	11653	0	0	6	0
ASV126	</td>	9231	0	985	5	39010
ASV127	</td>	8921	0	0	0	287
ASV128	</td>	6754	0	0	0	25
ASV129	</td>	2244	0	0	0	0
ASV13		256	45	271	7023	2962
ASV130	</td>	1907	0	0	0	0
ASV131	</td>	1635	0	0	0	0
ASV132	</td>	1520	0	0	0	0
ASV133		19	0	0	0	0
ASV134		11	0	0	0	3
ASV135		136	0	494	3695	1856
ASV136		6	0	0	0	0
ASV137		15	0	0	0	0
ASV138		3	0	0	0	0
ASV139		3	0	0	0	0
ASV140		4	0	0	0	0
ASV142	</td>	3	0	0	0	0
ASV143		12	0	0	0	0
ASV144		5	0	0	0	0
ASV145	</td>	4	0	0	0	0
ASV146	</td>	3	0	0	0	0
ASV147		3	0	0	0	0
ASV148		4	0	0	0	0
ASV149		2	0	0	0	0
ASV150	</td>	2	0	0	0	0
ASV151		63	0	22	848	385
ASV152		152	0	117	5351	1216
ASV153		151	0	275	2459	846
ASV154		135	0	92	3907	1534
ASV155		102	0	59	2478	738
ASV156		80	0	354	869	542
ASV157		75	0	87	2827	1293
ASV158		68	0	195	2330	543
ASV159		54	0	3581	919	2256
ASV160		43	0	19	924	568
ASV161		39	0	42	755	167
ASV162		41	0	0	1327	241
ASV164		42	0	506	682	1968
ASV165		25	0	18	689	196
ASV166		28	0	0	38	20
ASV168		33	0	0	506	109
ASV169		31	0	0	354	82
ASV17	</td>	566	64	400	16373	6024
ASV170		21	0	30	1081	342
ASV171		18	0	18	209	30
ASV172		19	0	18	899	259
ASV173		26	0	13	355	199
ASV174		17	0	34	675	296
ASV175		16	0	16	142	251
ASV176		25	0	311	332	85
ASV177		14	0	28	385	165
ASV178		16	0	0	484	92
ASV179		11	0	51	264	97
ASV180		12	0	0	156	10
ASV181		17	0	0	93	14
ASV182		11	0	462	127	0
ASV183		16	0	0	849	212
ASV184		8	0	0	55	0
ASV185		9	0	0	21	0
ASV186		10	0	663	256	24
ASV187		11	0	87	129	15
ASV188		5	0	0	0	0
ASV189		8	0	32	424	84
ASV190		7	0	0	73	0
ASV191		6	0	0	154	118
ASV192		2	0	0	0	0
ASV193		2	0	0	0	0
ASV195		5	0	0	13	2
ASV196		54	0	30	1260	240
ASV197		20	0	10	263	38
ASV198		10	0	9	135	73
ASV200		10	0	0	0	0
ASV202		12	0	476	270	101
ASV204		9	0	0	75	3
ASV205		5	0	9	27	32
ASV207		12	0	0	30	11
ASV209		6	0	0	121	13
ASV210		10	0	233	344	75
ASV212		28	0	1635	161	0
ASV213		15	0	0	405	421
ASV214		2	0	0	2	0
ASV215		7	0	0	0	0
ASV216		8	0	0	22	36
ASV217		32	0	66	1331	500
ASV219		14	0	1065	53	44
ASV22		106	8	75	2736	747
ASV220		8	0	0	0	0
ASV221		9	0	17	14	25
ASV222	</td>	6	0	0	81	42
ASV223		3	0	0	0	0
ASV224		4	0	165	0	0
ASV225	</td>	53	0	64	1543	1041
ASV226		5	0	0	160	50
ASV227		9	0	0	31	0
ASV228		10	0	14	129	27
ASV229		25	0	613	238	43
ASV23		251	21	250	7173	2615
ASV230		5	0	0	0	0
ASV231		6	0	15	368	26
ASV232		33	0	0	297	88
ASV233		46	0	3102	1710	279
ASV234		2	0	3	106	27
ASV235		14	0	77	183	45
ASV236		4	0	0	103	6
ASV237		3	0	0	0	0
ASV238		11	0	141	0	0
ASV239		9	0	0	97	38
ASV24		297	40	371	9958	4200
ASV240		9	0	0	23	6
ASV244		7	0	18	347	18
ASV246		31	0	37	1163	413
ASV247		3	0	0	46	0
ASV248		7	0	61	0	0
ASV25		333	22	293	9062	3716
ASV28		290	37	274	8453	4121
ASV4		192	70849	2893	1578750	3560
ASV43		69	6	36	1654	483
ASV44	</td>	51	4	25	1111	333
ASV59		30	2	15	828	308
ASV6	</td>	138	2903	5785	239901	1324
ASV67		78	4	775	1805	2560
ASV68		138	15	79	3058	946
ASV71		174	9	156	4212	1137
ASV72		39	6	0	1278	645
ASV73		15	2	7	200	50
ASV74		94	8	80	3096	884
ASV75		10	3	10	187	46
ASV76		93	5	114	2938	1080
ASV78		104	6	98	2712	672
ASV80		129	24	104	4487	1493
ASV81		369	23	396	11567	4121
ASV84		19	5	19	668	212
ASV96		25	8	17	610	240

</tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tr> </tbody> </table></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul></li> </ul>

The taxonomic classifications associated with the ASVs remaining in DNA standards after removal of contaminants. Expected member of DNA standard are demarked with *.

featureID	expected_member	Taxon
ASV10	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Enterococcaceae;g__Enterococcus;s__NA
ASV101		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV106		k__Bacteria;p__Deferribacteres;c__Deferribacteres;o__Deferribacterales;f__Deferribacteraceae;g__Mucispirillum;s__NA
ASV110		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV118		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__NA
ASV120		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV122	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Bacillaceae 1;g__Bacillus;s__Bacillus_licheniformis(CP000002)
ASV123	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_fermentum(NR_118978.1)
ASV124	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV125	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Listeriaceae;g__Listeria;s__NA
ASV126	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Escherichia/Shigella;s__NA
ASV127	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Salmonella;s__NA
ASV128	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Pseudomonadaceae;g__Pseudomonas;s__NA
ASV129	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV13		k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Bifidobacteriales;f__Bifidobacteriaceae;g__Bifidobacterium;s__NA
ASV130	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Bacillaceae 1;g__Bacillus;s__Bacillus_licheniformis(CP000002)
ASV131	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Escherichia/Shigella;s__NA
ASV132	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Salmonella;s__Salmonella_enterica(AE006468)
ASV133		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV134		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV135		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV136		k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV137		k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV138		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV139		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV140		k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV142	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV143		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV144		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV145	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_fermentum(JN175331)
ASV146	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Salmonella;s__Salmonella_enterica(AE006468)
ASV147		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV148		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__NA;s__NA
ASV149		k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV150	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Bacillaceae 1;g__Bacillus;s__NA
ASV151		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV152		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV153		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV154		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV155		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Stomatobaculum;s__NA
ASV156		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV157		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV158		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV159		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnospiracea incertae sedis;s__NA
ASV160		k__Bacteria;p__Proteobacteria;c__Deltaproteobacteria;o__Desulfovibrionales;f__Desulfovibrionaceae;g__NA;s__NA
ASV161		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV162		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV164		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Intestinimonas;s__NA
ASV165		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV166		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV168		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV169		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Clostridium IV;s__NA
ASV17	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_johnsonii(AJ002515)
ASV170		k__Bacteria;p__Deferribacteres;c__Deferribacteres;o__Deferribacterales;f__Deferribacteraceae;g__Mucispirillum;s__NA
ASV171		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__NA
ASV172		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV173		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Oscillibacter;s__Oscillibacter_valericigenes(AB238598)
ASV174		k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV175		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Alistipes;s__Alistipes_sp.(JF824799)
ASV176		k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Coriobacteriales;f__Coriobacteriaceae;g__NA;s__NA
ASV177		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Ruminococcus2;s__NA
ASV178		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV179		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV180		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV181		k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV182		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV183		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__Faecalitalea;s__NA
ASV184		k__Bacteria;p__Proteobacteria;c__Alphaproteobacteria;o__Rhodospirillales;f__Rhodospirillaceae;g__NA;s__NA
ASV185		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV186		k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA
ASV187		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__NA;s__NA
ASV188		k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV189		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV190		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV191		k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA
ASV192		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV193		k__Bacteria;p__Bacteroidetes;c__NA;o__NA;f__NA;g__NA;s__NA
ASV195		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV196		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV197		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Oscillibacter;s__NA
ASV198		k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA
ASV200		k__Bacteria;p__Proteobacteria;c__Deltaproteobacteria;o__Desulfovibrionales;f__Desulfovibrionaceae;g__Desulfovibrio;s__NA
ASV202		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV204		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV205		k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV207		k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Serratia;s__Serratia_plymuthica(AJ233433)
ASV209		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV210		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV212		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV213		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Oscillibacter;s__NA
ASV214		k__Bacteria;p__Verrucomicrobia;c__Opitutae;o__Puniceicoccales;f__Puniceicoccaceae;g__Coraliomargarita;s__NA
ASV215		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV216		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV217		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV219		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV22		k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Helicobacteraceae;g__Helicobacter;s__NA
ASV220		k__Bacteria;p__Deferribacteres;c__Deferribacteres;o__Deferribacterales;f__Deferribacteraceae;g__Mucispirillum;s__NA
ASV221		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__NA;s__NA
ASV222	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Pseudomonadaceae;g__Pseudomonas;s__NA
ASV223		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV224		k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA
ASV225	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__NA
ASV226		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV227		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Blautia;s__NA
ASV228		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV229		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV23		k__Bacteria;p__Tenericutes;c__Mollicutes;o__Anaeroplasmatales;f__Anaeroplasmataceae;g__Anaeroplasma;s__NA
ASV230		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Eubacteriaceae;g__Eubacterium;s__NA
ASV231		k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Coriobacteriales;f__Coriobacteriaceae;g__Enterorhabdus;s__NA
ASV232		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV233		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Clostridium XlVa;s__NA
ASV234		k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Actinomycetales;f__Propionibacteriaceae;g__Propionibacterium;s__NA
ASV235		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Flavonifractor;s__NA
ASV236		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV237		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV238		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV239		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Moryella;s__NA
ASV24		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV240		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV244		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Alistipes;s__NA
ASV246		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__NA
ASV247		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV248		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV25		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV28		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV4		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_caecimuris(NR_144606.1)
ASV43		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Alistipes;s__NA
ASV44	</td>	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Pseudomonadaceae;g__Pseudomonas;s__NA
ASV59		k__Bacteria;p__Proteobacteria;c__Deltaproteobacteria;o__NA;f__NA;g__NA;s__NA
ASV6	</td>	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__NA
ASV67		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_goldsteinii(AY974070)
ASV68		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV71		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_acidifaciens(AB021164)
ASV72		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV73		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV74		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV75		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV76		k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Bifidobacteriales;f__Bifidobacteriaceae;g__Bifidobacterium;s__NA
ASV78		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV80		k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV81		k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV84		k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV96		k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Helicobacteraceae;g__Helicobacter;s__NA

Contaminants identified for Nanopore. Total abundance for each ASV within the projects are shown.

featureID	CMS001	dna_std	GFU005	Nanopore	SPF
ASV101	291	62	796	6	0
ASV103	211	11	893	2	62
ASV106	870	95	2276	7	27
ASV107	3	0	550	7	0
ASV110	1594	113	4018	6	1570
ASV118	3949	383	11339	7	6831
ASV120	513	41	1500	5	61
ASV13	2962	256	7023	45	271
ASV17	6024	566	16373	64	400
ASV22	747	106	2736	8	75
ASV23	2615	251	7173	21	250
ASV24	4200	297	9958	40	371
ASV25	3716	333	9062	22	293
ASV28	4121	290	8453	37	274
ASV32	0	0	1045	12	0
ASV42	0	0	432	10	0
ASV43	483	69	1654	6	36
ASV44	333	51	1111	4	25
ASV46	0	0	129	3	0
ASV59	308	30	828	2	15
ASV62	0	0	106	2	0
ASV67	2560	78	1805	4	775
ASV68	946	138	3058	15	79
ASV70	321	16	1189	7	80352
ASV71	1137	174	4212	9	156
ASV72	645	39	1278	6	0
ASV73	50	15	200	2	7
ASV74	884	94	3096	8	80
ASV75	46	10	187	3	10
ASV76	1080	93	2938	5	114
ASV78	672	104	2712	6	98
ASV80	1493	129	4487	24	104
ASV81	4121	369	11567	23	396
ASV84	212	19	668	5	19
ASV96	240	25	610	8	17

The taxonomic classifications associated with the ASVs identified as likely cross-project contaminants for Nanopore.

featureID	Taxon
ASV101	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV103	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV106	k__Bacteria;p__Deferribacteres;c__Deferribacteres;o__Deferribacterales;f__Deferribacteraceae;g__Mucispirillum;s__NA
ASV107	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV110	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV118	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__NA
ASV120	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV13	k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Bifidobacteriales;f__Bifidobacteriaceae;g__Bifidobacterium;s__NA
ASV17	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_johnsonii(AJ002515)
ASV22	k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Helicobacteraceae;g__Helicobacter;s__NA
ASV23	k__Bacteria;p__Tenericutes;c__Mollicutes;o__Anaeroplasmatales;f__Anaeroplasmataceae;g__Anaeroplasma;s__NA
ASV24	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__NA;g__NA;s__NA
ASV25	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV28	k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV32	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV42	k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV43	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Alistipes;s__NA
ASV44	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Pseudomonadales;f__Pseudomonadaceae;g__Pseudomonas;s__NA
ASV46	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV59	k__Bacteria;p__Proteobacteria;c__Deltaproteobacteria;o__NA;f__NA;g__NA;s__NA
ASV62	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Clostridium IV;s__NA
ASV67	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_goldsteinii(AY974070)
ASV68	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV70	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_apodemi(AJ871178)
ASV71	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_acidifaciens(AB021164)
ASV72	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV73	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV74	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV75	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV76	k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Bifidobacteriales;f__Bifidobacteriaceae;g__Bifidobacterium;s__NA
ASV78	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV80	k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__NA;s__NA
ASV81	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV84	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV96	k__Bacteria;p__Proteobacteria;c__Epsilonproteobacteria;o__Campylobacterales;f__Helicobacteraceae;g__Helicobacter;s__NA

Contaminants identified for GFU005. Total abundance for each ASV within the projects are shown.

featureID	CMS001	dna_std	GFU005	Nanopore	SPF
ASV1123	2500	0	30	0	0
ASV122	11	15195	4	0	0
ASV123	0	13128	12	0	0
ASV1230	9332	0	22	0	0
ASV124	0	12401	3	0	0
ASV125	0	11653	6	0	0
ASV126	39010	9231	5	0	985
ASV1305	214546	0	19	0	0
ASV1332	40520	0	11	0	0
ASV1333	54823	0	8	0	0
ASV1337	18424	0	3	0	0
ASV1338	9128	0	3	0	0
ASV1339	81912	0	6	0	0
ASV1340	18528	0	7	0	0
ASV1341	128363	0	7	0	0
ASV1344	9029	0	2	0	0
ASV1346	37413	0	4	0	0
ASV1350	9904	0	9	0	0
ASV1351	112645	0	5	0	0
ASV1352	43089	0	7	0	0
ASV1472	3442	0	2	0	0
ASV1511	7440	0	2	0	0
ASV1658	6105	0	2	0	0
ASV1667	22869	0	4	0	0
ASV1679	203522	0	10	0	0
ASV1680	246420	0	6	0	0
ASV1681	31292	0	11	0	0
ASV243	31093	68	460	0	11037
ASV286	5219	0	2	0	0
ASV289	2002	0	3	0	0
ASV290	17	0	3	0	891
ASV291	32411	0	3	0	0
ASV489	2432	0	27	0	0
ASV503	0	0	13	0	6879
ASV645	0	0	35	0	7106
ASV650	11650	0	4	0	0
ASV670	40	0	44	0	3356
ASV690	22	0	9	0	1972
ASV724	10949	0	3	0	282
ASV739	185	0	5	0	883
ASV794	0	0	12	0	1073
ASV819	175084	0	15	0	5
ASV913	5677	0	17	0	93

The taxonomic classifications associated with the ASVs identified as likely cross-project contaminants for GFU005.

featureID	Taxon
ASV1123	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Alistipes;s__Alistipes_indistinctus(AB490804)
ASV122	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Bacillaceae 1;g__Bacillus;s__Bacillus_licheniformis(CP000002)
ASV123	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Lactobacillaceae;g__Lactobacillus;s__Lactobacillus_fermentum(NR_118978.1)
ASV1230	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV124	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV125	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Listeriaceae;g__Listeria;s__NA
ASV126	k__Bacteria;p__Proteobacteria;c__Gammaproteobacteria;o__Enterobacteriales;f__Enterobacteriaceae;g__Escherichia/Shigella;s__NA
ASV1305	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_dorei(AB242142)
ASV1332	k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Coriobacteriales;f__Coriobacteriaceae;g__Collinsella;s__NA
ASV1333	k__Bacteria;p__Firmicutes;c__Negativicutes;o__Selenomonadales;f__Acidaminococcaceae;g__Phascolarctobacterium;s__Phascolarctobacterium_faecium(X72865)
ASV1337	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV1338	k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Coriobacteriales;f__Coriobacteriaceae;g__NA;s__NA
ASV1339	k__Bacteria;p__Actinobacteria;c__Actinobacteria;o__Bifidobacteriales;f__Bifidobacteriaceae;g__Bifidobacterium;s__Bifidobacterium_adolescentis(AP009256)
ASV1340	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__NA
ASV1341	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Pseudobutyrivibrio;s__Pseudobutyrivibrio_ruminis(X95893)
ASV1344	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Sutterella;s__NA
ASV1346	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Clostridium IV;s__NA
ASV1350	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Ruminococcus;s__NA
ASV1351	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnospiracea incertae sedis;s__NA
ASV1352	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__NA
ASV1472	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__Streptococcus_salivarius(AY188354)
ASV1511	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Eubacteriaceae;g__Eubacterium;s__NA
ASV1658	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__NA
ASV1667	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Oscillibacter;s__NA
ASV1679	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_vulgatus(CP000139)
ASV1680	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides;s__Bacteroides_uniformis(AB050110)
ASV1681	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnospira;s__NA
ASV243	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Clostridium XlVa;s__NA
ASV286	k__Bacteria;p__Firmicutes;c__Negativicutes;o__Selenomonadales;f__Veillonellaceae;g__Dialister;s__NA
ASV289	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__NA
ASV290	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV291	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__NA
ASV489	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV503	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV645	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV650	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Prevotellaceae;g__Prevotella;s__NA
ASV670	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV690	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV724	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV739	k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Streptococcus;s__NA
ASV794	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV819	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Faecalibacterium;s__Faecalibacterium_prausnitzii(AJ413954)
ASV913	k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA

Contaminants identified for SPF. Total abundance for each ASV within the projects are shown.

featureID	CMS001	dna_std	GFU005	Nanopore	SPF
ASV1	1784	0	713676	33359	30
ASV106	870	95	2276	7	27
ASV141	17364	81	2303	0	163
ASV167	4788	27	538	0	12
ASV19	47	0	20689	1045	262
ASV2	107465	0	1834129	51383	53
ASV20	56	0	68425	3143	176
ASV201	16075	17	545	0	4
ASV242	3796	17	156	0	17
ASV287	189	0	317	0	4
ASV325	0	0	318	0	2
ASV347	0	0	415	0	5
ASV463	56	0	127	0	2
ASV79	0	0	26099	1784	35
ASV798	127085	0	15	0	6
ASV8	75	0	55147	3654	70
ASV813	13292	0	0	0	4
ASV819	175084	0	15	0	5
ASV828	14433	0	0	0	2
ASV9	116	0	27288	1785	64
ASV913	5677	0	17	0	93

The taxonomic classifications associated with the ASVs identified as likely cross-project contaminants for SPF.

featureID	Taxon
ASV1	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Clostridium XlVa;s__NA
ASV106	k__Bacteria;p__Deferribacteres;c__Deferribacteres;o__Deferribacterales;f__Deferribacteraceae;g__Mucispirillum;s__NA
ASV141	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV167	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__Lachnospiracea incertae sedis;s__NA
ASV19	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV2	k__Bacteria;p__Verrucomicrobia;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Verrucomicrobiaceae;g__Akkermansia;s__Akkermansia_muciniphila(AY271254)
ASV20	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV201	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_distasonis(AB238922)
ASV242	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Parabacteroides;s__Parabacteroides_distasonis(AB238922)
ASV287	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV325	k__Bacteria;p__Cyanobacteria/Chloroplast;c__Chloroplast;o__-;f__Chloroplast;g__Streptophyta;s__NA
ASV347	k__NA;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV463	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Comamonadaceae;g__Delftia;s__NA
ASV79	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Clostridium IV;s__NA
ASV798	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Gemmiger;s__NA
ASV8	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV813	k__Bacteria;p__Firmicutes;c__Erysipelotrichia;o__Erysipelotrichales;f__Erysipelotrichaceae;g__Holdemanella;s__NA
ASV819	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Faecalibacterium;s__Faecalibacterium_prausnitzii(AJ413954)
ASV828	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV9	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV913	k__Bacteria;p__Firmicutes;c__NA;o__NA;f__NA;g__NA;s__NA

Contaminants identified for CMS001. Total abundance for each ASV within the projects are shown.

featureID	CMS001	dna_std	GFU005	Nanopore	SPF
ASV107	3	0	550	7	0
ASV122	11	15195	4	0	0
ASV1343	2	0	63	0	0
ASV15	3	0	228	11	0
ASV218	6	4	36	0	1466
ASV290	17	0	3	0	891
ASV357	3	0	123	0	0
ASV468	99	0	222	0	3116
ASV470	39	0	100	0	2668
ASV664	65	0	126	0	3060
ASV670	40	0	44	0	3356
ASV680	12	0	112	0	992
ASV681	28	0	0	0	1615
ASV686	17	0	0	0	526
ASV690	22	0	9	0	1972
ASV7	38	0	153030	9782	498
ASV707	2	0	0	0	1589
ASV718	4	0	12	0	518
ASV721	2	0	53	0	295
ASV723	2	0	30	0	171
ASV8	75	0	55147	3654	70
ASV9	116	0	27288	1785	64

The taxonomic classifications associated with the ASVs identified as likely cross-project contaminants for CMS001.

featureID	Taxon
ASV107	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV122	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Bacillaceae 1;g__Bacillus;s__Bacillus_licheniformis(CP000002)
ASV1343	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV15	k__Bacteria;p__Verrucomicrobia;c__Verrucomicrobiae;o__Verrucomicrobiales;f__Verrucomicrobiaceae;g__Akkermansia;s__Akkermansia_muciniphila(AY271254)
ASV218	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Anaerotruncus;s__NA
ASV290	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV357	k__Bacteria;p__Tenericutes;c__Mollicutes;o__Anaeroplasmatales;f__Anaeroplasmataceae;g__Anaeroplasma;s__NA
ASV468	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV470	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV664	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Oscillibacter;s__NA
ASV670	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV680	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV681	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__NA;s__NA
ASV686	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__NA;g__NA;s__NA
ASV690	k__Bacteria;p__NA;c__NA;o__NA;f__NA;g__NA;s__NA
ASV7	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV707	k__Bacteria;p__Firmicutes;c__Bacilli;o__Bacillales;f__Staphylococcaceae;g__Staphylococcus;s__NA
ASV718	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__NA;s__NA
ASV721	k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Porphyromonadaceae;g__Barnesiella;s__NA
ASV723	k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Ruminococcaceae;g__Clostridium III;s__NA
ASV8	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA
ASV9	k__Bacteria;p__Proteobacteria;c__Betaproteobacteria;o__Burkholderiales;f__Sutterellaceae;g__Parasutterella;s__NA

</div>

Supplementary Information

Theoretical composition of culture and DNA controls. Adapted from Zymo manual for products 6300, 6305

Species	Theoretical Commposition (%)
Pseudomonas aeruginosa	4.2
Enterococcus faecalis	9.9
Escherichia coli	10.1
Salmonella enterica	10.4
Listeria monocytogenes	14.1
Staphylococcus aureus	15.5
Bacillus subtilis	17.4
Lactobacillus fermentum	18.4

Members of MM12, in GFU005 and Nanopore

Species
Clostridium innocuum
Bacteroides caecimuris
Lactobacillus reuterii
Enterococcus faecalis
Actualibacter muris
Bifidobacterium animalis
Muribaculum intestinale
Flavonifractor plautii
Clostridium clostriodiforme
Akkermansia muciniphila
Turicimonas muris
Blautia coccoides

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