Hello. I have a question regarding the make.contigs syntax.
The sequence I got using iSeq equipment is 151bp.
However, if you make forward and reverse contigs with the make.contigs syntax, a total sequence of nearly 300bp is created.
I expected it to be around 151bp.
I wonder why.
Please review.
Thank you.
Good question, I need to think before I answer
Itâs likely that your amplicons do not share any meaningful overlap, so make.contigs joined the forward and reverse reads using whatever it could find in common at the ends of the sequences. The âoverlapâ may be as short as a single bp. This can happen if the maximum read length of the sequencer (2x 150 bp) is shorter than the targeted region. Basically, your contigs might be junk. The forward (or reverse) reads could still be useful on their own though.
Can you share more information about your project? Like what is the overall research objective (metagenomics? gene profiling?), and what is the target region?
Hello. Thuja.
Thank you for your kind reply.
Currently, I am analyzing two types of dust samples in the environment and samples of Tsetse fly from Africa.
I want to check the distribution of bacteria in two types of samples.
At the same time, I would also like to identify eukaryotic biota by amplifying the 18S V9 region.
As you said, we have confirmed that the reverse sequences are actually combined with the foreword in reverse complementarity.
So does that mean itâs pointless to build a contig from the amplicons I have?
I want to create a contig by applying de novo assembly, but what cannot use this method?
If we cannot use this method, it is difficult to identify the level from the current silva database to species, so we plan to conduct analysis using NCBIâs ânrâ database.
Thank you for your response.
Could you post the command line you are using? And the logfile, if possible.
Hello. Leocadia.
Thank you so much for showing interest in my article.
I think itâs a little long, but I uploaded the whole log once.
In my opinion, we will proceed with âDe novo assemplyâ with the final file âstability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fastaâ analyzed by mothur.
I want to get the exact species name by applying the contigs thus obtained to the NCBI ânrâ database.
Is it possible?
Please review.
Thank you.
Windows version
mothur v.1.45.3
Last updated: 5/8/21
by
Patrick D. Schloss
[NOTE]: Setting random seed to 19760620.
Interactive Mode
mothur >
align.seqs(fasta=C.tuberculostearicum.16S.V4.fasta, reference=silva.nr_v138_1.align)
Using 4 processors.
Reading in the silva.nr_v138_1.align template sequences... DONE.
It took 492 to read 146601 sequences.
Aligning sequences from C.tuberculostearicum.16S.V4.fasta ...
Reducing processors to 1.
It took 1 secs to align 1 sequences.
It took 12 seconds to align 1 sequences.
Output File Names:
C.tuberculostearicum.16S.V4.align
C.tuberculostearicum.16S.V4.align.report
mothur >
summary.seqs(fasta=C.tuberculostearicum.16S.V4.align)
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 11895 25318 293 0 4 1
2.5%-tile: 11895 25318 293 0 4 1
25%-tile: 11895 25318 293 0 4 1
Median: 11895 25318 293 0 4 1
75%-tile: 11895 25318 293 0 4 1
97.5%-tile: 11895 25318 293 0 4 1
Maximum: 11895 25318 293 0 4 1
Mean: 11895 25318 293 0 4
# of Seqs: 1
It took 0 secs to summarize 1 sequences.
Output File Names:
C.tuberculostearicum.16S.V4.summary
mothur >
pcr.seqs(fasta=silva.nr_v138_1.align, start=11895, end=25318, keepdots=F, processors=8)
Using 8 processors.
[NOTE]: no sequences were bad, removing silva.nr_v138_1.bad.accnos
It took 460 secs to screen 146601 sequences.
Output File Names:
silva.nr_v138_1.pcr.align
mothur >
rename.file(input=silva.nr_v138_1.pcr.align, new=silva.v4.fasta)
Current files saved by mothur:
fasta=silva.nr_v138_1.pcr.align
processors=8
summary=C.tuberculostearicum.16S.V4.summary
mothur >
make.file(inputdir=., type=gz, prefix=stability)
Output File Names:
stability.files
mothur >
make.contigs(file=stability.files, processors=8)
Using 8 processors.
Group count:
Mâ51 101721
Mâ52 93826
Mâ53 134029
Mâ55 164051
Mâ62 136451
Mâ65 114612
Mâ06 86802
Mâ07 90197
Mâ09 128188
Mâ14 165063
Mâ26 129797
Total of all groups is 1344737
It took 534 secs to process 1344737 sequences.
Output File Names:
stability.trim.contigs.fasta
stability.scrap.contigs.fasta
stability.contigs.report
stability.contigs.groups
mothur >
summary.seqs(fasta=stability.trim.contigs.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 146 146 0 3 1
2.5%-tile: 1 223 223 0 11 33619
25%-tile: 1 279 279 0 13 336185
Median: 1 281 281 0 14 672369
75%-tile: 1 297 297 3 57 1008553
97.5%-tile: 1 298 298 14 59 1311119
Maximum: 1 302 302 56 118 1344737
Mean: 1 283 283 2 26
# of Seqs: 1344737
It took 48 secs to summarize 1344737 sequences.
Output File Names:
stability.trim.contigs.summary
mothur >
screen.seqs(fasta=stability.trim.contigs.fasta, group=stability.contigs.groups, maxambig=0, maxlength=300)
Using 8 processors.
It took 35 secs to screen 1344737 sequences, removed 407523.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.bad.accnos.temp, group=stability.contigs.groups)
Removed 407523 sequences from your group file.
Output File Names:
stability.contigs.pick.groups
/******************************************/
Output File Names:
stability.trim.contigs.good.fasta
stability.trim.contigs.bad.accnos
stability.contigs.good.groups
It took 71 secs to screen 1344737 sequences.
mothur >
summary.seqs(fasta=stability.trim.contigs.good.fasta)
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 148 148 0 11 1
2.5%-tile: 1 275 275 0 11 23431
25%-tile: 1 280 280 0 13 234304
Median: 1 295 295 0 15 468608
75%-tile: 1 298 298 0 57 702911
97.5%-tile: 1 298 298 0 60 913784
Maximum: 1 300 300 0 110 937214
Mean: 1 287 287 0 32
# of Seqs: 937214
It took 28 secs to summarize 937214 sequences.
Output File Names:
stability.trim.contigs.good.summary
mothur >
unique.seqs(fasta=stability.trim.contigs.good.fasta)
937214 914376
Output File Names:
stability.trim.contigs.good.names
stability.trim.contigs.good.unique.fasta
mothur >
count.seqs(name=stability.trim.contigs.good.names, group=stability.contigs.good.groups)
It took 30 secs to create a table for 937214 sequences.
Total number of sequences: 937214
Output File Names:
stability.trim.contigs.good.count_table
mothur >
summary.seqs(count=stability.trim.contigs.good.count_table)
Using stability.trim.contigs.good.unique.fasta as input file for the fasta parameter.
Using 8 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 148 148 0 11 1
2.5%-tile: 1 275 275 0 11 23431
25%-tile: 1 280 280 0 13 234304
Median: 1 295 295 0 15 468608
75%-tile: 1 298 298 0 57 702911
97.5%-tile: 1 298 298 0 60 913784
Maximum: 1 300 300 0 110 937214
Mean: 1 287 287 0 32
# of unique seqs: 914376
total # of seqs: 937214
It took 38 secs to summarize 937214 sequences.
Output File Names:
stability.trim.contigs.good.unique.summary
mothur >
align.seqs(fasta= stability.trim.contigs.good.unique.fasta, reference=silva.v4.fasta, flip=t)
Using 4 processors.
Reading in the silva.v4.fasta template sequences... DONE.
It took 75 to read 146601 sequences.
Aligning sequences from stability.trim.contigs.good.unique.fasta ...
It took 3763 secs to align 914376 sequences.
[WARNING]: 422435 of your sequences generated alignments that eliminated too many bases, a list is provided in stability.trim.contigs.good.unique.flip.accnos.
[NOTE]: 163103 of your sequences were reversed to produce a better alignment.
It took 3770 seconds to align 914376 sequences.
Output File Names:
stability.trim.contigs.good.unique.align
stability.trim.contigs.good.unique.align.report
stability.trim.contigs.good.unique.flip.accnos
mothur >
summary.seqs(fasta=silva.v4.fasta)
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 9875 83 0 3 1
2.5%-tile: 2 13423 289 0 3 3666
25%-tile: 2 13423 291 0 4 36651
Median: 2 13423 291 0 5 73301
75%-tile: 2 13423 291 0 5 109951
97.5%-tile: 2 13423 458 1 6 142936
Maximum: 4226 13423 1519 5 16 146601
Mean: 2 13422 308 0 4
# of Seqs: 146601
It took 72 secs to summarize 146601 sequences.
Output File Names:
silva.v4.summary
mothur >
screen.seqs(fasta=stability.trim.contigs.good.unique.align, count=stability.trim.contigs.good.count_table, start= 2, end= 13423, maxhomop=16)
Using 4 processors.
It took 249 secs to screen 914376 sequences, removed 886183.
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.bad.accnos.temp, count=stability.trim.contigs.good.count_table)
Removed 908602 sequences from your count file.
Output File Names:
stability.trim.contigs.good.pick.count_table
/******************************************/
Output File Names:
stability.trim.contigs.good.unique.good.align
stability.trim.contigs.good.unique.bad.accnos
stability.trim.contigs.good.good.count_table
It took 514 secs to screen 914376 sequences.
mothur >
summary.seqs(fasta=current, count=current)
Using stability.trim.contigs.good.good.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.align as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 13423 261 0 11 1
2.5%-tile: 1 13423 295 0 11 716
25%-tile: 2 13423 297 0 13 7154
Median: 2 13423 297 0 13 14307
75%-tile: 2 13423 297 0 14 21460
97.5%-tile: 2 13423 298 0 15 27897
Maximum: 2 13423 300 0 16 28612
Mean: 1 13423 296 0 13
# of unique seqs: 28193
total # of seqs: 28612
It took 14 secs to summarize 28612 sequences.
Output File Names:
stability.trim.contigs.good.unique.good.summary
mothur >
filter.seqs(fasta=stability.trim.contigs.good.unique.good.align,vertical=T,trump=.)
Using 4 processors.
Creating Filter...
It took 8 secs to create filter for 28193 sequences.
Running Filter...
It took 7 secs to filter 28193 sequences.
Length of filtered alignment: 545
Number of columns removed: 12878
Length of the original alignment: 13423
Number of sequences used to construct filter: 28193
Output File Names:
stability.filter
stability.trim.contigs.good.unique.good.filter.fasta
mothur >
unique.seqs(fasta=stability.trim.contigs.good.unique.good.filter.fasta, count= stability.trim.contigs.good.good.count_table)
28193 28192
Output File Names:
stability.trim.contigs.good.unique.good.filter.count_table
stability.trim.contigs.good.unique.good.filter.unique.fasta
mothur >
pre.cluster(fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, count=stability.trim.contigs.good.unique.good.filter.count_table, diffs=2)
Using 4 processors.
/******************************************/
Running command: split.groups(groups=Mâ51-Mâ52-Mâ53-Mâ55-Mâ62-Mâ65-Mâ06-Mâ07-Mâ09-Mâ14-Mâ26, fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, count=stability.trim.contigs.good.unique.good.filter.count_table)
Using 4 processors.
/******************************************/
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ53.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ53)
/******************************************/
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ51.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ51)
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ65.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ65)
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ09.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ09)
Selected 332 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ53
/******************************************/
Done.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ55.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ55)
Selected 2827 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ51
Selected 3118 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ09
/******************************************/
Done.
Selected 3615 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ65
/******************************************/
Done.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ52.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ52)
/******************************************/
Selected 2567 sequences from your fasta file.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ14.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ14)
/******************************************/
Done.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ52.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ52)
/******************************************/
Selected 2567 sequences from your fasta file.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ06.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ06)
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ55
/******************************************/
Done.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ62.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ62)
Selected 2706 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ52
/******************************************/
Done.
Selected 2496 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ06
/******************************************/
Done.
Selected 2845 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ14
/******************************************/
Done.
Selected 2814 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ62
/******************************************/
Done.
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ07.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ07)
/******************************************/
Running command: get.seqs(dups=f, accnos=stability.trim.contigs.good.unique.good.filter.Mâ26.count_table.accnos, fasta=stability.trim.contigs.good.unique.good.filter.unique.fastaMâ26)
Selected 2430 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ07
/******************************************/
Done.
Selected 2751 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fastaMâ26
/******************************************/
Done.
Output File Names:
stability.trim.contigs.good.unique.good.filter.Mâ51.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ51.fasta
stability.trim.contigs.good.unique.good.filter.Mâ52.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ52.fasta
stability.trim.contigs.good.unique.good.filter.Mâ53.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ53.fasta
stability.trim.contigs.good.unique.good.filter.Mâ55.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ55.fasta
stability.trim.contigs.good.unique.good.filter.Mâ62.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ62.fasta
stability.trim.contigs.good.unique.good.filter.Mâ65.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ65.fasta
stability.trim.contigs.good.unique.good.filter.Mâ06.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ06.fasta
stability.trim.contigs.good.unique.good.filter.Mâ07.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ07.fasta
stability.trim.contigs.good.unique.good.filter.Mâ09.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ09.fasta
stability.trim.contigs.good.unique.good.filter.Mâ14.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ14.fasta
stability.trim.contigs.good.unique.good.filter.Mâ26.count_table
stability.trim.contigs.good.unique.good.filter.unique.Mâ26.fasta
/******************************************/
Processing group Mâ53:
Processing group Mâ65:
Mâ53 332 332 0
Processing group Mâ09:
Total number of sequences before pre.cluster was 332.
pre.cluster removed 0 sequences.
It took 0 secs to cluster 332 sequences.
Processing group Mâ55:
Processing group Mâ51:
Mâ55 2567 2548 19
Total number of sequences before pre.cluster was 2567.
pre.cluster removed 19 sequences.
It took 1 secs to cluster 2567 sequences.
Processing group Mâ62:
Mâ51 2827 2827 0
Total number of sequences before pre.cluster was 2827.
pre.cluster removed 0 sequences.
It took 1 secs to cluster 2827 sequences.
Processing group Mâ52:
Mâ09 3118 3099 19
Total number of sequences before pre.cluster was 3118.
pre.cluster removed 19 sequences.
It took 1 secs to cluster 3118 sequences.
Processing group Mâ14:
Mâ65 3615 3584 31
Total number of sequences before pre.cluster was 3615.
pre.cluster removed 31 sequences.
It took 1 secs to cluster 3615 sequences.
Processing group Mâ06:
Mâ62 2814 2803 11
Mâ52 2706 2705 1
Total number of sequences before pre.cluster was 2814.
Total number of sequences before pre.cluster was 2706.
pre.cluster removed 11 sequences.
pre.cluster removed 1 sequences.
It took 0 secs to cluster 2814 sequences.
It took 0 secs to cluster 2706 sequences.
Mâ14 2845 2817 28
Mâ06 2496 2487 9
Total number of sequences before pre.cluster was 2496.
pre.cluster removed 9 sequences.
Total number of sequences before pre.cluster was 2845.
pre.cluster removed 28 sequences.
It took 0 secs to cluster 2496 sequences.
Processing group Mâ07:
It took 0 secs to cluster 2845 sequences.
Processing group Mâ26:
Mâ07 2430 2416 14
Total number of sequences before pre.cluster was 2430.
pre.cluster removed 14 sequences.
It took 1 secs to cluster 2430 sequences.
Mâ26 2751 2731 20
Total number of sequences before pre.cluster was 2751.
pre.cluster removed 20 sequences.
It took 1 secs to cluster 2751 sequences.
Deconvoluting count table results...
It took 0 secs to merge 28349 sequences group data.
/******************************************/
Running command: get.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.fasta, accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table.temp)
Selected 28073 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.pick.fasta
/******************************************/
It took 13 secs to run pre.cluster.
Using 4 processors.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ51.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ52.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ53.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ55.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ62.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ65.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ06.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ07.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ09.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ14.map
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ26.map
mothur >
chimera.uchime(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.count_table, dereplicate=t)
Using 4 processors.
[DEBUG]: uchime location using D:\mothur_Tsetse_silva_v138_1_V4_210925\uchime.exe
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ51.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ51.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ52.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ52.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ53.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ53.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ55.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ55.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ62.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ62.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ65.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ65.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ06.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ06.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ07.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ07.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ09.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ09.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ14.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ14.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ26.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.Mâ26.fasta
/******************************************/
It took 2 secs to check 332 sequences from group Mâ53.
It took 110 secs to check 2548 sequences from group Mâ55.
It took 117 secs to check 2827 sequences from group Mâ51.
It took 151 secs to check 3099 sequences from group Mâ09.
It took 205 secs to check 3584 sequences from group Mâ65.
It took 120 secs to check 2705 sequences from group Mâ52.
It took 126 secs to check 2803 sequences from group Mâ62.
It took 113 secs to check 2817 sequences from group Mâ14.
It took 80 secs to check 2487 sequences from group Mâ06.
It took 66 secs to check 2731 sequences from group Mâ26.
It took 50 secs to check 2416 sequences from group Mâ07.
It took 335 secs to check 28349 sequences.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.chimeras
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.accnos
mothur >
remove.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.fasta, accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.accnos)
Removed 15 sequences from your fasta file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta
mothur >
summary.seqs(fasta=current)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta as input file for the fasta parameter.
Using 4 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 545 261 0 11 1
2.5%-tile: 1 545 295 0 11 702
25%-tile: 1 545 297 0 13 7015
Median: 1 545 297 0 13 14030
75%-tile: 1 545 297 0 14 21044
97.5%-tile: 1 545 298 0 15 27357
Maximum: 1 545 300 0 16 28058
Mean: 1 545 296 0 13
# of Seqs: 28058
It took 1 secs to summarize 28058 sequences.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.summary
mothur >
classify.seqs(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.count_table, reference=silva.seed_v138_1.align, taxonomy=silva.seed_v138_1.tax)
Using 4 processors.
Generating search database... DONE.
It took 21 seconds generate search database.
Reading in the silva.seed_v138_1.tax taxonomy... DONE.
Calculating template taxonomy tree... DONE.
Calculating template probabilities... DONE.
It took 39 seconds get probabilities.
Classifying sequences from stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta ...
[WARNING]: FS10001784_2_BRB11601-2529_1_1110_10900_1340 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1114_10570_3530 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1105_8300_2860 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1112_3350_1570 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1112_3210_2000 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1113_12690_1820 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1104_14980_2220 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1102_10060_2120 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1106_13570_1290 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
[WARNING]: FS10001784_2_BRB11601-2529_1_1110_5770_3210 could not be classified. You can use the remove.lineage command with taxon=unknown; to remove such sequences.
**** Exceeded maximum allowed command warnings, silencing warnings ****
It took 160 secs to classify 28058 sequences.
It took 2 secs to create the summary file for 28058 sequences.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.tax.summary
mothur >
remove.lineage(fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.count_table, taxonomy= stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.taxonomy, taxon=Chloroplast-Mitochondria-unknown-Archaea-Eukaryota-unclassified)
/******************************************/
Running command: remove.seqs(accnos=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.accnos, count=stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.count_table, fasta=stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.fasta)
Removed 20 sequences from your fasta file.
Removed 20 sequences from your count file.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.pick.count_table
/******************************************/
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.pick.taxonomy
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.accnos
stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.pick.count_table
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.fasta
mothur >
summary.tax(taxonomy=current, count=current)
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.denovo.uchime.pick.pick.count_table as input file for the count parameter.
Using stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.pick.taxonomy as input file for the taxonomy parameter.
It took 1 secs to create the summary file for 28574 sequences.
Output File Names:
stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.seed_v138_1.wang.pick.tax.summary
I think there is some confusion there. What you have there is V4 (according to your file), 16S V4 (prokaryota), and that one I think is about 300 bp.
the 18S v9 is for sure about 131 bp in metazoans (what you are looking in the 150 bp?). But, that does not give species resolution. Genus if you are lucky, in general gives family-level resolution.
But, for your original question (âHowever, if you make forward and reverse contigs with the make.contigs syntax, a total sequence of nearly 300bp is created.I expected it to be around 151bp.â) we would need the logfile of that step. But, I think you might be mixing 18V9 and 16SV4.
To back up leocadio, The V4 region with primers and barcodes will be about 290 bp. Regardless, youâre going to get minimal overlap between the 151 nt reads (about 10 nt?). The V4 region without barcodes and primers is right around 250 nt long. You really. need fully overlapping sequence reads like you would get with 2x250 nt reads of the V4 region. If you donât youâll get very high sequencing error rates for the assembled reads.
Also, it is impossible to reliably obtain species level taxonomies from a 16S or 18S rRNA gene sequence. Anyone telling you otherwise is stretching things. You would need genome sequences to get that level of resolution. Just because you see a species name on a sequence in the nr database, doesnât mean that you can get that level of resolution for an unknown, short sequence.
Pat
Sorry to confuse you. Leocadia.
The âMothurâ log I posted is for 16S V4.
18S V9 is not uploaded.
Thank you for your kind explanation. pschloss. Thanks again for answering my basic question. ^^
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