去除质体和非细菌

最后发布时间 : 2022-11-04 11:23:23 浏览量 :

R脚本选择细菌古菌(真核)、去除叶绿体、线粒体并统计比例；输出筛选并排序的OTU表
输入为OTU表result/raw/otutab.txt和物种注释result/raw/otus.sintax
输出筛选并排序的特征表result/otutab.txt和
统计污染比例文件result/raw/otutab_nonBac.txt和过滤细节otus.sintax.discard
真菌ITS数据，请改用otutab_filter_nonFungi.R脚本，只筛选真菌

过滤特征表

otutab.txt

#OTU ID	KO1	KO2	KO3	KO4	KO5	KO6	OE1	OE2	OE3	OE4	OE5	OE6	WT1	WT2	WT3	WT4	WT5	WT6
ASV_1	382	671	282	438	381	390	476	590	500	360	490	357	799	830	567	664	508	519
ASV_10	107	129	231	264	372	240	70	65	82	105	138	103	138	201	83	94	189	93
ASV_100	22	34	4	6	10	22	43	20	27	23	23	38	19	12	28	16	22	27
ASV_1000	1	1	3	0	0	2	1	1	3	1	2	0	0	0	1	1	4	3
ASV_1001	3	1	0	0	0	3	3	3	0	1	1	2	1	0	3	4	0	1
ASV_1002	4	1	1	4	4	2	5	2	2	5	3	7	6	7	3	5	0	1

otus.sintax

ASV_13	d:Bacteria(1.00),p:Bacteroidetes(1.00),c:Flavobacteriia(1.00),o:Flavobacteriales(1.00),f:Flavobacteriaceae(1.00),g:Flavobacterium(1.00)	+	d:Bacteria,p:Bacteroidetes,c:Flavobacteriia,o:Flavobacteriales,f:Flavobacteriaceae,g:Flavobacterium
ASV_17	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Piscinibacter(0.32)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Piscinibacter
ASV_7	d:Bacteria(1.00),p:Bacteroidetes(1.00),c:Flavobacteriia(1.00),o:Flavobacteriales(1.00),f:Flavobacteriaceae(1.00),g:Flavobacterium(1.00)	+	d:Bacteria,p:Bacteroidetes,c:Flavobacteriia,o:Flavobacteriales,f:Flavobacteriaceae,g:Flavobacterium
ASV_5	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Chloroplast(1.00),f:Chloroplast(1.00),g:Streptophyta(1.00)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Chloroplast,f:Chloroplast,g:Streptophyta
ASV_1	d:Bacteria(1.00),p:Actinobacteria(1.00),c:Actinobacteria(1.00),o:Streptosporangiales(0.85),f:Thermomonosporaceae(0.80),g:Actinocorallia(0.48)	+	d:Bacteria,p:Actinobacteria,c:Actinobacteria,o:Streptosporangiales,f:Thermomonosporaceae,g:Actinocorallia
ASV_2	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Pelomonas(1.00)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Pelomonas
ASV_3	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Rhizobacter(0.92)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Rhizobacter

Rscript script/otutab_filter_nonBac.R \
      --input results/matrix/otutab.txt \
      --taxonomy results/matrix/otus.sintax \
      --output results/otutab.txt\
      --stat results/matrix/otutab_nonBac.stat \
      --discard results/matrix/otus.sintax.discard

otutab.txt

#OTUID	KO1	KO2	KO3	KO4	KO5	KO6	OE1	OE2	OE3	OE4	OE5	OE6	WT1	WT2	WT3	WT4	WT5	WT6
ASV_1	382	671	282	438	381	390	476	590	500	360	490	357	799	830	567	664	508	519
ASV_2	671	412	863	793	1032	631	245	169	414	440	348	239	440	536	267	386	497	424
ASV_3	213	173	369	296	445	308	211	151	372	367	316	242	327	635	357	416	413	274
ASV_4	506	141	181	257	480	178	203	196	134	210	214	129	362	473	196	192	394	290
ASV_8	189	195	215	148	77	101	132	183	540	300	211	190	230	275	199	356	169	184
ASV_6	302	232	307	309	490	365	89	95	114	143	134	137	178	191	154	184	158	158

otutab_nonBac.stat

SampleID	total_reads	nonspecific_reads	chloroplast_reads	filtered_reads
KO1	11355	8	17	11330
KO2	12473	4	52	12417
KO3	13494	1	74	13419
KO4	13171	1	13	13157
KO5	13847	0	61	13786
KO6	12598	1	76	12521
OE1	11992	14	513	11465
OE2	11738	6	431	11301
OE3	11956	6	282	11668
OE4	11799	10	143	11646
OE5	12692	4	629	12059
OE6	11585	5	552	11028
WT1	13162	9	520	12633
WT2	13389	11	450	12928
WT3	12370	10	372	11988
WT4	12807	12	325	12470
WT5	13303	10	592	12701
WT6	12615	11	170	12434

otus.sintax.discard

ASV_944	d:Eukaryota(0.68),p:Plantae(0.68),c:Liliopsida(0.68),o:Poales(0.68),f:Poaceae(0.68),g:Zea(0.68)	+	d:Eukaryota,p:Plantae,c:Liliopsida,o:Poales,f:Poaceae,g:Zea
ASV_1428	NA	NA	NA
ASV_5	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Chloroplast(1.00),f:Chloroplast(1.00),g:Streptophyta(1.00)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Chloroplast,f:Chloroplast,g:Streptophyta
ASV_137	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Cyanobacteria(1.00),f:Family_IX(0.01),g:GpIX(0.01)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Cyanobacteria
ASV_131	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Cyanobacteria(1.00),f:Family_I(0.82),g:Potamolinea(0.08)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Cyanobacteria,f:Family_I
ASV_597	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Cyanobacteria(1.00),f:Family_I(1.00),g:Desikacharya(0.58)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Cyanobacteria,f:Family_I,g:Desikacharya
ASV_978	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Chloroplast(1.00),f:Chloroplast(1.00),g:Streptophyta(1.00)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Chloroplast,f:Chloroplast,g:Streptophyta
ASV_979	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(0.97),c:Cyanobacteria(0.84),f:Family_XIII(0.12),g:Cephalothrix(0.07)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Cyanobacteria,f:Family_XIII
ASV_1147	d:Bacteria(1.00),p:Cyanobacteria/Chloroplast(1.00),c:Cyanobacteria(1.00),f:Family_I(0.68),g:Potamolinea(0.14)	+	d:Bacteria,p:Cyanobacteria/Chloroplast,c:Cyanobacteria,f:Family_I,g:Potamolinea

过滤特征表对应序列

cut -f 1 results/otutab.txt | tail -n+2 > results/otutab.id
usearch -fastx_getseqs results/raw/otus.fa \
    -labels results/otutab.id -fastaout results/otus.fa

otus.fa

>ASV_1
GTAGTCCACGCCGTAAACGGTGGGCGCTAGATGTGGGGACCTTCCACGGTTTCTGCGTCGCAGCTAACGCATTAAGCGCC
CCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTA
ATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATCGCCGGAAAACTCGCAGAGATGCGGGGTCCTTTTGGGCCGG
TGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCT
ATGTTGCCAGCACGCCCTTCGGGGTGGTGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGA
>ASV_2
GTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAGGGTTTCTTCTCAGTAACGTAGCTAACGCGTGAAGTTGACC
GCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAAT
TCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCTGGAATCCTGAAGAGATTTGGGAGTGCTCGAAAGAGAGCC
AGAACACAGGTGCTGCATGGCCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGT
CATTAGTTGCTACGAAAGGGCACTCTAATGAGACTGCCGGTGACAAACCGGA

过滤特征表对应序列注释

awk 'NR==FNR{a[$1]=$0}NR>FNR{print a[$1]}'\
    results/matrix/otus.sintax results/otutab.id \
    > results/otus.sintax

otus.sintax

ASV_1	d:Bacteria(1.00),p:Actinobacteria(1.00),c:Actinobacteria(1.00),o:Streptosporangiales(0.85),f:Thermomonosporaceae(0.80),g:Actinocorallia(0.48)	+	d:Bacteria,p:Actinobacteria,c:Actinobacteria,o:Streptosporangiales,f:Thermomonosporaceae,g:Actinocorallia
ASV_2	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Pelomonas(1.00)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Pelomonas
ASV_3	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Rhizobacter(0.92)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Rhizobacter
ASV_4	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(1.00),g:Rhizobacter(1.00)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Rhizobacter
ASV_8	d:Bacteria(1.00),p:Actinobacteria(1.00),c:Actinobacteria(1.00),o:Streptomycetales(1.00),f:Streptomycetaceae(1.00),g:Streptomyces(0.99)	+	d:Bacteria,p:Actinobacteria,c:Actinobacteria,o:Streptomycetales,f:Streptomycetaceae,g:Streptomyces
ASV_6	d:Bacteria(1.00),p:Proteobacteria(1.00),c:Betaproteobacteria(1.00),o:Burkholderiales(1.00),f:Comamonadaceae(0.93),g:Piscinibacter(0.28)	+	d:Bacteria,p:Proteobacteria,c:Betaproteobacteria,o:Burkholderiales,f:Comamonadaceae,g:Piscinibacter
ASV_7	d:Bacteria(1.00),p:Bacteroidetes(1.00),c:Flavobacteriia(1.00),o:Flavobacteriales(1.00),f:Flavobacteriaceae(1.00),g:Flavobacterium(1.00)	+	d:Bacteria,p:Bacteroidetes,c:Flavobacteriia,o:Flavobacteriales,f:Flavobacteriaceae,g:Flavobacterium

统计OTU

usearch -otutab_stats results/otutab.txt \
    -output results/otutab.stat

otutab.stat

  220951  Reads (221.0k)
        18  Samples
      1521  OTUs

     27378  Counts
      5170  Count  =0  (18.9%)
      4891  Count  =1  (17.9%)
      4017  Count >=10 (14.7%)

       437  OTUs found in all samples (28.7%)
       609  OTUs found in 90% of samples (40.0%)
      1426  OTUs found in 50% of samples (93.8%)

Sample sizes: min 11028, lo 11646, med 12434, mean 12275.1, hi 12701, max 13786

此时的得到以下文件用于后续分析

results/otutab.txt
results/otus.fa
results/otus.sintax

物种注释物种注释分类汇总

test

2022-11-06 23:06:31

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