1 1

2 2
2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.1 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.2 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3 5
3.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2 OTU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.2.2 Rarefaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.2.3 Shannon-Wiener . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2.4 Simpson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2.5 OTU Venn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.4 α . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.5 ANOSIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.6 β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.6.1 PCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.6.2 PCoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.6.3 NMDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.7.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.7.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.7.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.7.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.12.1 KEGG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.12.2 COG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

28

29
1. 1 of 29

LYS 56 16S V4

13983895620
/
[email protected]

17750629984
[email protected]
[email protected]
2. 2 of 29

2.1

DNA DNA

DNA

2.1.1 DNA
DNA
DNA

2.1.2 DNA
DNA DNA
DNA DNA DNA 2
1 Multiskan™ GO DNA DNA
2 DNA

2.1.3
16S rDNA V3-V4
341F 806R 16S V3-V4
515F 806R 16S V4 Illumina 16S Qubit 3.0
pooling
2. 3 of 29

2.1.4

3
1 Qubit 3.0
2 Agilent 2100 DNA
3 Q-PCR

2.1.5
pooling flowcell Illumina
2. 4 of 29

2.2

LEFSe
OTU Shannon Anosim ( 5 ) ( )
Chao ANOVA ( 3 )
Simpson RDA ( ) ( )
Shannon PCA CCA ( )
PCoA
Simpson
Heatmap
3. 5 of 29

3.1

FASTQ read 4 @ ID
N + reads
@HISEQ15:146:C6VAJANXX:7:1101:6695:1978 1:N:0:TCATTC
ATCGGAAGAGCACACGTCTGAACTCCAGTCACTCATTCATCTCGTATGCCGTCTT
+
=<ABFGGGGGGGGGGGEGEFGGGGGGDGEFGG@GGFFGGF1BFFEFEGGGGGGG

3.1: Reads reads

, , Phred quality score
, 20 99%, reads ,
,
3. 6 of 29

3.2 OTU

3.2.1
Illumina Hiseq/MiniSeq Raw reads Flash
[1] Clean reads Chimera_check QIIME [2]
OTU OTU 3.1

3.1:

Sample Raw Clean Chimera_check OTUseq OTU

A10D10 38473 38226 34509 34495 126

A10P10 37059 36809 32430 32419 111
A1D1 46121 45760 41321 41298 83
A1P1 38895 38630 34055 34052 95
A2D2 32235 32008 31163 31163 53
A2P2 42861 42569 40882 40877 56
A3D3 80267 79673 72050 71998 78
A3P3 112084 109232 100818 100792 102
A4D4 37935 37633 32415 32397 237
A4P4 46036 45598 38409 38391 227
A5D5 39874 39576 37075 36974 90
A5P5 32103 31881 30570 30544 101
A6D6 65559 64925 59669 59657 95
A6P6 36298 35917 32727 32646 82
A7D7 41487 41110 35523 35515 96
A7P7 42781 42399 37271 37269 84
A8D8 54137 53716 47848 47834 218
A8P8 42091 41656 37182 37167 187
A9D9 39661 39406 36729 36548 91

1 Raw reads ,Clean reads , Flash;Chimera_check

;OTUseq OTU ,OTU OTU
2 report/2_OTU_assembly_analysis/2_2_assemble_table/ .xls
3. 7 of 29

3.2.2 Rarefaction

OTU
Qiime [2]

3.2: (Rarefaction Curve) OTU

3. 8 of 29

3.2.3 Shannon-Wiener
Shannon-Wiener Shannon-Wiener
Shannon-Wiener

3.3: Shannon-Wiener Shannon-Wiener

Shannon-Wiener

Shannon-wiener
∑
S obs
ni ni
HShannon = − ln �
i=1
N N

, Sobs OTU ni i OTU N

3. 9 of 29

3.2.4 Simpson
Simpson Simpson

3.4: Simpson Simpson

Simpson

Simpson
∑Sobs
i=1 ni (ni − 1)
Dsimpson = �
N (N − 1)
, Sobs OTU ni i OTU N
3. 10 of 29

3.2.5 OTU Venn

OTU OTU
5

3.5: OTU OTU

3. 11 of 29

3.3

3.6: 20 ,
3. 12 of 29

3.4 α

α α
ACE Chao1 Observed species Shannon Simpson J

3.7: Alpha alpha report/group*/

4alpha_diversity .xls
3. 13 of 29

3.5 ANOSIM

ANOSIM
Bray-Curtis

3.8: ANOSIM (Between) Between

R∈(-1 1) R>0 R<0
P P< 0.05
3. 14 of 29

3.6 β

β OTU
PCA Principal
Component Analysis PCoA Principal Co-ordinates Analysis NMDS Non-
Metric Multi-Dimensional Scaling PCA OTU PCoA NMDS UniFrac
OTU OTU

3.6.1 PCA
PCA

3.9: PCA , ,
PCA PC1
PC2
3. 15 of 29

3.6.2 PCoA
PCoA PCA PCA
PCoA PCoA
UniFracs Euclidean, Bray-Curtis Jaccard UniFracs R Phyloseq [3]

3.10: PCoA , ,
.PCoA
PC1 2 PC2
3. 16 of 29

3.6.3 NMDS
NMDS MDS
PCoA NMDS

NMDS

3.11: NMDS
3. 17 of 29

3.7

3.7.1
OTU 20
correlation Bray-Curtis

3.12: 20 ,
3. 18 of 29

3.7.2
3.7.1

3.13: 3.12 , 20 , ,
,
3. 19 of 29

3.7.3

3.14: ,
3. 20 of 29

3.7.4

3.15: 3.14 , ,
3. 21 of 29

3.8

,
GraPhlAn [4]

3.16: , (
); 15 (A-O) ( 6 ),
, ,
3. 22 of 29

3.9

Statistical hypothesis testing

Wilcox Kruskal-Wallis

3.17: 10
3. 23 of 29

3.10

10 Pearson

3.18: 10
(p<0.001:***; p<0.01:**; p<0.05:*)
3. 24 of 29

3.11

LEfSe [5] LDA Effect Size , ,

, :
(1) Kruskal-Wallis ; (2)
, Wilcoxon ; (3) Linear Discriminant
Analysis (LDA) ( LDA score)

3.19: LDA , LDA Score , biomaker

,
3. 25 of 29

3.20:

3.21: biomaker . biomaker 1

biomaker
3. 26 of 29

3.12

16S , ,
PICRUSt [6] 16S
, 16S
95%, COG KEGG [7]

3.12.1 KEGG
KEGG ,

3.22: KEGG kegg ( ) ,

,error bar ; P
3. 27 of 29

3.12.2 COG
PICRUSt COG , COG

3.23: COG COG ( ) ,

,error bar ; P
3. 28 of 29

/
0_raw_reads
0_1_base_quality| *_quality.png [ reads ]
1_clean_reads
1_1_base_quality| *_quality.png [ ]
2_OTU_assembly_analysis
2_1_species_distribution� *.svg;*xls[ ]
2_2_assemble_table�sample2sequence_summary.xls [reads otu ]
2_2_assemble_table�otus.fa [otu ]
2_2_assemble_table�*.tre [ ]
LEfSe_sample_file.xls [LEfSe ]
otu_tax_table_norm.xls [ otu ]
otu_tax_table.xls [ otu ]
3_rarefaction
*.svg [ ]
group*
4_alpha_diversity
richness_plot.svg|multi_adiversity.xls [ alpha ]
5_Anosim_analysis
anosim_plot.svg|otu_table_norm.xls [Anosim ]
6_beta_diversity
6_1_ordinatioin� pca_plot.svg [PCA ]
6_1_ordinatioin� PCoA_plot.svg [PCoA ]
6_1_ordinatioin� NMDS_plot.svg [NMDS ]
6_2_heatmap
6_2_1_with_clust� *.svg [ heatmap ]
6_2_2_without_clust� *.svg [ heatmap ]
*.xls [ heatmap ]
6_3_clust� *.svg *xls [ ]
7_environment_factor
7_1_CCA� cca_plot.svg [CCA ]
7_2_RDA� rda_plot.svg [RDA ]
8_species_visulization
speciese_distribution_group.svg [ ]
9_differential_species_analysis
9_1_LEFSe_analysis
otu_table_lefse_LDA.svg [LEFSe LDA ]
otu_table_lefse_cladogram.svg [LEFSe ]
differential_species_distribution [LEFSe ]
9_2_diff_analysis| *.svg [ ]
9_3_corr_analysis| *.svg [ ]
10_function_analysis
10_1_KEGG� KEGG_pathway.svg [KEGG ]
10_1_KEGG� kegg_pathway_prediction_count.xls [KEGG ]
10_2_COG� COG_function.svg [COG ]
10_2_COG� cog_prediction_count.xls [COG ]
picrust_otus_css.biom [ PICRUSt ]
Linux Windows excel Linux less
svg AI png,jpg,tiff Windows Linux
EditPlus
 29 of 29

[1] Tanja Magoč and Steven L. Salzberg. Flash: fast length adjustment of short reads to improve genome assemblies. Bioinformatics,
27(21):2957–2963, 2011.

[2] J. G. Caporaso, J Kuczynski, J Stombaugh, K Bittinger, F. D. Bushman, E. K. Costello, N Fierer, A. G. Peña, J. K. Goodrich, and
J. I. Gordon. QIIME allows analysis of high-throughput community sequencing data. Nature Methods, 7(5):335, 2010.

[3] P. J. Mcmurdie and S Holmes. phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data.
Plos One, 8(4):e61217, 2013.

[4] Francesco Asnicar, George Weingart, Timothy L. Tickle, Curtis Huttenhower, and Nicola Segata. Compact graphical representation of
phylogenetic data and metadata with GraPhlAn. 3(11):e1029, 2015.

[5] Joseph N Paulson, O Colin Stine, Héctor Corrada Bravo, and Mihai Pop. Differential abundance analysis for microbial marker-gene
surveys. Nature Methods, 10(12):1200, 2013.

[6] Morgan G I Langille, Jesse Zaneveld, J Gregory Caporaso, Daniel Mcdonald, Knights Dan, Joshua A Reyes, Jose C Clemente, Deron E
Burkepile, Rebecca L Vega Thurber, and Rob Knight. Predictive functional profiling of microbial communities using 16s rRNA marker
gene sequences. Nature Biotechnology, 31(9):814, 2013.

[7] M Kanehisa, M Araki, S Goto, M Hattori, M Hirakawa, M Itoh, T Katayama, S Kawashima, S Okuda, and T Tokimatsu. KEGG for
linking genomes to life and the environment. Nucleic Acids Research, 36(Database issue):D480–D484, 2008.