Hi
I would like to understand why the difference between the total sequence numbers on three outputs files after classify.seqs.
Could you please explain why the numbers are different? What do they mean? Why do the number of seques reduce?
And could you explain which file (values) are more accurate to describe the microbial diversity?
I send the headers of the out put files bellow, and also the commands the provided them.
Thank you for any help
kind regards
Igor
File 1:
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tax.summary
taxonomy total A B C D
Root 54669 2681 1353 29338 21297
File 2:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.tax.summary
taxonomy total A B C D
Root 941 195 148 376 398
File 3:
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.1.cons.tax.summary
taxonomy total A B C D
Root 205 98 72 68 117
log file:
Windows version
Running 64Bit Version
mothur v.1.39.5
Last updated: 3/20/2017
by
Patrick D. Schloss
Department of Microbiology & Immunology
University of Michigan
http://www.mothur.org
When using, please cite:
Schloss, P.D., et al., Introducing mothur: Open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol, 2009. 75(23):7537-41.
Distributed under the GNU General Public License
Type ‘help()’ for information on the commands that are available
For questions and analysis support, please visit our forum at https://www.mothur.org/forum
Type ‘quit()’ to exit program
Batch Mode
mothur > screen.seqs(fasta=Merged.fasta, group=merge.groups, maxambig=0, processors=2)
Using 2 processors.
Output File Names:
Merged.good.fasta
Merged.bad.accnos
merge.good.groups
It took 3 secs to screen 262721 sequences.
mothur > unique.seqs()
Using Merged.good.fasta as input file for the fasta parameter.
262721 19462
Output File Names:
Merged.good.names
Merged.good.unique.fasta
mothur > count.seqs(name=current, group=current) Using merge.good.groups as input file for the group parameter. Using Merged.good.names as input file for the name parameter.
Using 2 processors.
It took 1 secs to create a table for 262721 sequences.
Total number of sequences: 262721
Output File Names:
Merged.good.count_table
mothur > align.seqs(fasta=current, reference=silva.nr_v128.align) Using Merged.good.unique.fasta as input file for the fasta parameter.
Using 2 processors.
Reading in the silva.nr_v128.align template sequences… DONE.
It took 220 to read 190661 sequences.
Aligning sequences from Merged.good.unique.fasta …
Reading in the silva.nr_v128.align template sequences… DONE.
It took 544 to read 190661 sequences.
[WARNING]: Some of your sequences generated alignments that eliminated too many bases, a list is provided in Merged.good.unique.flip.accnos. If you set the flip parameter to true mothur will try aligning the reverse compliment as well.
It took 867 secs to align 19462 sequences.
Output File Names: Merged.good.unique.align Merged.good.unique.align.report Merged.good.unique.flip.accnos
mothur > summary.seqs(fasta=current, count=current) Using Merged.good.count_table as input file for the count parameter. Using Merged.good.unique.align as input file for the fasta parameter.
Using 2 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 0 0 0 0 1 1
2.5%-tile: 23439 35461 329 0 5 6569
25%-tile: 23439 35461 333 0 5 65681
Median: 23439 35461 334 0 5 131361
75%-tile: 23439 35461 334 0 5 197041
97.5%-tile: 23439 35461 334 0 5 256153
Maximum: 43116 43116 552 0 16 262721
Mean: 23436.6 35452.4 333.379 0 4.9997
of unique seqs: 19462
total # of seqs: 262721
Output File Names:
Merged.good.unique.summary
It took 16 secs to summarize 262721 sequences.
mothur > get.current()
Current RAM usage: 7.76687 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
fasta=Merged.good.unique.align
group=merge.good.groups
name=Merged.good.names
count=Merged.good.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > screen.seqs(fasta=current, count=current, maxhomop=10, start=23439, end=35461) Using Merged.good.count_table as input file for the count parameter. Using Merged.good.unique.align as input file for the fasta parameter.
Using 2 processors.
Output File Names:
Merged.good.unique.good.align
Merged.good.unique.bad.accnos
Merged.good.good.count_table
It took 18 secs to screen 19462 sequences.
mothur > get.current()
Current RAM usage: 8.65634 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
fasta=Merged.good.unique.good.align
group=merge.good.groups
name=Merged.good.names
count=Merged.good.good.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > filter.seqs(fasta=current, vertical=T, trump=.) Using Merged.good.unique.good.align as input file for the fasta parameter.
Using 2 processors.
Creating Filter…
Running Filter...
Length of filtered alignment: 625 Number of columns removed: 49375 Length of the original alignment: 50000 Number of sequences used to construct filter: 17932
Output File Names:
Merged.filter
Merged.good.unique.good.filter.fasta
mothur > get.current()
Current RAM usage: 7.9452 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
fasta=Merged.good.unique.good.filter.fasta
group=merge.good.groups
name=Merged.good.names
count=Merged.good.good.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > unique.seqs(fasta=current, count=current) Using Merged.good.good.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.fasta as input file for the fasta parameter. 17932 17664
Output File Names:
Merged.good.unique.good.filter.count_table
Merged.good.unique.good.filter.unique.fasta
mothur > get.current()
Current RAM usage: 7.96848 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
fasta=Merged.good.unique.good.filter.unique.fasta
group=merge.good.groups
name=Merged.good.names
count=Merged.good.unique.good.filter.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > pre.cluster(fasta=current, count=current, diffs=4) Using Merged.good.unique.good.filter.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.fasta as input file for the fasta parameter.
Using 2 processors.
Processing group A:
Processing group C:
5725 907 4818
Total number of sequences before pre.cluster was 5725.
pre.cluster removed 4818 sequences.
It took 0 secs to cluster 5725 sequences.
Processing group D:
5586 1211 4375
Total number of sequences before pre.cluster was 5586.
pre.cluster removed 4375 sequences.
It took 0 secs to cluster 5586 sequences.
Processing group B:
6915 1114 5801
Total number of sequences before pre.cluster was 6915.
pre.cluster removed 5801 sequences.
It took 1 secs to cluster 6915 sequences.
5209 1052 4157
Total number of sequences before pre.cluster was 5209.
pre.cluster removed 4157 sequences.
It took 1 secs to cluster 5209 sequences.
It took 1 secs to run pre.cluster.
Output File Names:
Merged.good.unique.good.filter.unique.precluster.fasta
Merged.good.unique.good.filter.unique.precluster.count_table
Merged.good.unique.good.filter.unique.precluster.A.map
Merged.good.unique.good.filter.unique.precluster.B.map
Merged.good.unique.good.filter.unique.precluster.C.map
Merged.good.unique.good.filter.unique.precluster.D.map
mothur > get.current()
Current RAM usage: 8.06574 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
fasta=Merged.good.unique.good.filter.unique.precluster.fasta
group=merge.good.groups
name=Merged.good.names
count=Merged.good.unique.good.filter.unique.precluster.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > chimera.vsearch(fasta=current, count=current, dereplicate=t) Using Merged.good.unique.good.filter.unique.precluster.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter.
Using 2 processors.
Checking sequences from Merged.good.unique.good.filter.unique.precluster.fasta …
It took 2 secs to check 1211 sequences from group A.
It took 2 secs to check 1052 sequences from group B.
It took 1 secs to check 907 sequences from group C.
It took 2 secs to check 1114 sequences from group D.
Output File Names:
Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table
Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.chimeras
Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
mothur > get.current()
Current RAM usage: 8.02036 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
fasta=Merged.good.unique.good.filter.unique.precluster.fasta
group=merge.good.groups
name=Merged.good.names
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > remove.seqs(fasta=current, accnos=current) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos as input file for the accnos parameter. Using Merged.good.unique.good.filter.unique.precluster.fasta as input file for the fasta parameter. [WARNING]: This command can take a namefile and you did not provide one. The current namefile is Merged.good.names which seems to match Merged.good.unique.good.filter.unique.precluster.fasta. Removed 355 sequences from your fasta file.
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.fasta
mothur > get.current()
Current RAM usage: 8.0202 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
fasta=Merged.good.unique.good.filter.unique.precluster.pick.fasta
group=merge.good.groups
name=Merged.good.names
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > classify.seqs(fasta=current, count=current, reference=silva.nr_v128.align, taxonomy=silva.nr_v128.tax, cutoff=70, output=simple, printlevel=7) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.fasta as input file for the fasta parameter.
Using 2 processors.
Generating search database… DONE.
It took 352 seconds generate search database.
Reading in the silva.nr_v128.tax taxonomy… DONE.
Calculating template taxonomy tree… DONE.
Calculating template probabilities… DONE.
It took 542 seconds get probabilities.
Classifying sequences from Merged.good.unique.good.filter.unique.precluster.pick.fasta …
Reading template taxonomy… DONE.
Reading template probabilities… [WARNING]: M_M03868_18_000000000-BBH4D_1_2119_6055_21907 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2119_5760_22769 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2113_14280_11147 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1105_18682_14953 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1114_26817_21514 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_8983_2118 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2110_3454_6502 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
DONE.
It took 82 seconds get probabilities.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1103_22773_3355 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2107_9159_19506 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2119_5810_21817 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1108_20049_21372 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1107_20352_18371 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1107_9844_15806 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2116_11256_4590 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1106_9671_24056 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1102_20493_19069 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1105_29415_11361 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1113_29115_11222 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2105_3750_19303 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_19907_21026 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_25504_22117 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1116_24135_7812 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
It took 180 secs to classify 3807 sequences.
[WARNING]: Your printlevel is greater than your maxlevel, adjusting your printlevel to 6
It took 0 secs to create the summary file for 3807 sequences.
Output File Names: Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.taxonomy Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.tax.summary
mothur > get.current()
Current RAM usage: 9.24657 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
fasta=Merged.good.unique.good.filter.unique.precluster.pick.fasta
group=merge.good.groups
name=Merged.good.names
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > remove.lineage(fasta=current, count=current, taxonomy=current, taxon=Cyanobacteria-Chloroplast-Mitochondria-Eukaryota) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.fasta as input file for the fasta parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.taxonomy as input file for the taxonomy parameter.
[NOTE]: The count file should contain only unique names, so mothur assumes your fasta, list and taxonomy files also contain only uniques.
Output File Names: Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.pick.taxonomy Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
mothur > get.current()
Current RAM usage: 9.24695 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
name=Merged.good.names
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.nr_v128.wang.pick.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > classify.seqs(fasta=current, count=current, reference=silva.nr_v128.align, taxonomy=silva.nr_v128.tax, cutoff=70, output=simple, printlevel=7) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta as input file for the fasta parameter.
Using 2 processors.
Reading template taxonomy… DONE.
Reading template probabilities… DONE.
It took 79 seconds get probabilities.
Classifying sequences from Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta …
Reading template taxonomy… DONE.
Reading template probabilities… [WARNING]: M_M03868_18_000000000-BBH4D_1_1114_26817_21514 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1109_20240_7750 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_8983_2118 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2110_3454_6502 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1103_22773_3355 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1108_20049_21372 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1107_9844_15806 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
DONE.
It took 80 seconds get probabilities.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2107_9159_19506 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2119_5810_21817 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2116_11256_4590 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1106_9671_24056 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1102_20493_19069 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1119_24329_2484 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1113_29115_11222 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2105_3750_19303 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2101_10666_8408 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_19907_21026 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2103_25504_22117 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2109_22642_11958 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_1116_24135_7812 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2119_6055_21907 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2119_5760_22769 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: M_M03868_18_000000000-BBH4D_1_2113_14280_11147 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
It took 117 secs to classify 1393 sequences.
[WARNING]: Your printlevel is greater than your maxlevel, adjusting your printlevel to 6
It took 0 secs to create the summary file for 1393 sequences.
Output File Names: Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tax.summary
mothur > get.current()
Current RAM usage: 9.25304 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
name=Merged.good.names
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > dist.seqs(fasta=current, cutoff=0.03) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta as input file for the fasta parameter.
Using 2 processors.
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
It took 1 seconds to calculate the distances for 1393 sequences.
mothur > get.current()
Current RAM usage: 9.25282 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
name=Merged.good.names
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > cluster(column=current, count=current, cutoff=0.03) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.dist as input file for the column parameter. Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter.
Using 2 processors.
Clustering Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
iter time label num_otus cutoff tp tn fp fn sensitivity specificity ppv npv fdr accuracy mcc f1score 0 0 0.03 1393 0.03 0 962447 0 7081 0 1 0 0.992696 0 0.992696 0 0 1 0 0.03 939 0.03 4978 961833 614 2103 0.703008 0.999362 0.8902 0.997818 0.1098 0.997198 0.789758 0.785607 2 0 0.03 941 0.03 5163 961792 655 1918 0.729134 0.999319 0.887418 0.99801 0.112582 0.997346 0.803115 0.800527 3 0 0.03 941 0.03 5160 961799 648 1921 0.728711 0.999327 0.88843 0.998007 0.11157 0.99735 0.803342 0.800683 4 0 0.03 941 0.03 5160 961799 648 1921 0.728711 0.999327 0.88843 0.998007 0.11157 0.99735 0.803342 0.800683
It took 0 seconds to cluster
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.steps
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.sensspec
mothur > get.current()
Current RAM usage: 9.25277 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > make.shared(list=current, count=current, label=0.03) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list as input file for the list parameter. 0.03
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
mothur > get.current()
Current RAM usage: 9.25262 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > classify.otu(list=current, count=current, taxonomy=current, label=0.03, output=simple) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list as input file for the list parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy as input file for the taxonomy parameter. 0.03 941
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.tax.summary
mothur > get.current()
Current RAM usage: 9.2861 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > count.groups(shared=current) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared as input file for the shared parameter. A contains 2681. B contains 1353. C contains 29338. D contains 21297.
Total seqs: 54669.
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.count.summary
mothur > get.current()
Current RAM usage: 9.28613 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > rarefaction.single(shared=current, calc=sobs, freq=100) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared as input file for the shared parameter.
Using 2 processors.
Processing group A
0.03
Processing group B
0.03
Processing group C
0.03
Processing group D
0.03
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.groups.rarefaction
mothur > get.current()
Current RAM usage: 9.29636 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > summary.single(shared=current, calc=nseqs-coverage-sobs-invsimpson) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared as input file for the shared parameter.
Processing group A
0.03
Processing group B
0.03
Processing group C
0.03
Processing group D
0.03
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.groups.summary
mothur > get.current()
Current RAM usage: 9.29635 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.list
name=Merged.good.names
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > phylotype(taxonomy=current) Using Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy as input file for the taxonomy parameter. [WARNING]: This command can take a namefile and you did not provide one. The current namefile is Merged.good.names which seems to match Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy. 1 2 3 4 5 6
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.sabund
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.rabund
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list
mothur > get.current()
Current RAM usage: 9.2967 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list
name=Merged.good.names
rabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.rabund
sabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.sabund
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > make.shared(list=current, count=current, label=1) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list as input file for the list parameter. 1
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.shared
mothur > get.current()
Current RAM usage: 9.29679 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list
name=Merged.good.names
rabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.rabund
sabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.sabund
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.opti_mcc.0.03.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > classify.otu(list=current, count=current, taxonomy=current, label=1, output=simple) Using Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table as input file for the count parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list as input file for the list parameter. Using Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy as input file for the taxonomy parameter. 1 205
Output File Names:
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.1.cons.taxonomy
Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.1.cons.tax.summary
mothur > get.current()
Current RAM usage: 9.30657 Gigabytes. Total Ram: 31.6925 Gigabytes.
Current files saved by mothur:
accnos=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.accnos
column=Merged.good.unique.good.filter.unique.precluster.pick.pick.dist
fasta=Merged.good.unique.good.filter.unique.precluster.pick.pick.fasta
group=merge.good.groups
list=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.list
name=Merged.good.names
rabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.rabund
sabund=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.sabund
shared=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.shared
taxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.taxonomy
constaxonomy=Merged.good.unique.good.filter.unique.precluster.pick.pick.nr_v128.wang.tx.1.cons.taxonomy
count=Merged.good.unique.good.filter.unique.precluster.denovo.vsearch.pick.pick.count_table
processors=2
summary=Merged.good.unique.summary
Current default directory saved by mothur: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Current working directory: D:\ALL_Metadata_usar_este\Kiwis_Vera\Kiwis\Kiwis_novosPrimers_cloroplastos\collapseq_fastq\
Output File Names:
current_files.summary
mothur > quit()
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>> Detected 50 [WARNING] messages, please review. <<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>>>>>>>