Have you followed the instructions from earlier in the thread? What are you still not able to do?
I’m unable to demultiplex without merging the paired reads (in mothur, the idemp tool suggested earlier works).
What I’m trying to get is one set of demultiplexed fastqs for the forwards reads, and another set for the reverse reads. Not a demultiplexed set of merged reads.
Sorry, but you’re going to have to use a different tool then.
Hi,
I am trying to work from the initial instructions on these feed; however, when I run make.contigs I keep getting this error:
mothur > make.contigs(ffastq=/home/Documents/data/R1.fastq, rfastq=/home/Documents/data/R2.fastq, rindex=/home/Documents/data/index.fastq, oligos=/home/Documents/data/my.oligos.txt)
rindex is not a valid parameter.
The valid parameters are: ffastq, rfastq, ffasta, rfasta, fqfile, rqfile, file, oligos, pdiffs, bdiffs, tdiffs, align, allfiles, trimoverlap, match, mismatch, gapopen, gapextend, insert, deltaq, processors, format, inputdir, and outputdir.
How is rindex not a valid parameter now, but it is still on the wiki? Is anyone else running into the issue or knows the solution?
Thanks!
What version of mothur are you using? When you start mothur, can you tell us what version number is at the top of the screen?
Pat
Now I am at the point of make.sra for NCBI, and I’m wondering if I had to follow Pat’s advice earlier in this thread and use the reverse complement of my barcodes, should I specify orientation=reverse for make.sra?
Thanks!
Beth
I am running Mothur v.1.37.6 with a Windows 7 PC.
I have just received paired end 16S mi-seq reads from Argonne and I am trying to make.contigs and demiltiplex the data. I received three files, the forward reads in one R1 file, reverse reads in one R2 file, and the index file (I1), with the sequences still contain the barcode but not the primers.
The first step I took was to create the reverse compliment for my oligos file. I followed Pat’s instructions using the reverseComp function in R. I then tried to run this oligos file with my data. My oligos file had “barcode” in the first column, the forward reading barcode in the second column, the reverse compliment barcode generated from the reverseComp in the third column, and the sample ID in the fourth column. First two lines are below:
barcode TAGGACGGGAGT TAGGACGGGAGT GoM_8045
barcode AAGTCTTATCTC AAGTCTTATCTC GoM_8046
Then I ran the following command:
mothur >
make.contigs(ffastq=Run1_16S_R1_001.fastq, rfastq=Run1_16S_R2_001.fastq, findex=Run1_16S_I1_001.fastq, oligos=Run1_16S_reverse_oligos.txt)
Using 1 processors.
Making contigs…
Done.
It took 22175 secs to process 16688339 sequences.
However, my groups file was empty and when I ran summary.seqs, it said my number of sequences had almost doubled, 32,930,404.
mothur >
summary.seqs()
Using Run1_16S_R1_001.trim.contigs.fasta as input file for the fasta parameter.
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 243 243 0 3 1
2.5%-tile: 1 252 252 0 4 823261
25%-tile: 1 253 253 0 4 8232602
Median: 1 253 253 1 4 16465203
75%-tile: 1 253 253 1 5 24697804
97.5%-tile: 1 377 377 15 6 32107144
Maximum: 1 502 502 115 251 32930404
Mean: 1 260.384 260.384 1.95789 4.62585
I feel my mistake is in my oligos file format and my make.contigs command. Is there a solution to this?
Can you post this as a new thread? FWIW, I think you might want to use NONE in place of the forward barcodes.
barcode NONE TAGGACGGGAGT GoM_8045
barcode NONE AAGTCTTATCTC GoM_8046
Hello Pat and Mothur team,
My case is a little bit different from the other folks that I could see on this thread.
I have 26 pairs of fastq files and a different combination of barcodes in each one. Only one barcode combination within each R1-R2 pair is of my interest. The rest is from other projects that I am not involved with.
The following is my oligo file:
############################
$ cat oligos-map4mothur.tab
primer CAGCMGCCGCGGTAATWC CCGTCAATTCCTTTRAGGTT 519F926R
BARCODE ggtac aggaa BfleaM1
BARCODE ggtac gagact NC014M10
BARCODE ggtac cgattcc FOF012M11
BARCODE ggtac tctcaatc DS006M12
BARCODE ggtac gagtgg XfleaM2
BARCODE ggtac ccacgtc FfleaM3
BARCODE ggtac ttctcagc SfleaM4
BARCODE ggtac ctagg NOfleaM5
BARCODE ggtac tgctta SAC023M6
BARCODE ggtac gcgaagt FOF007M7
BARCODE ggtac aatcctat DS024M8
BARCODE ggtac atctg SAC004M9
BARCODE caacac aggaa SAC015M13
BARCODE caacac gagact NC036M22
BARCODE caacac cgattcc SAC001M23
BARCODE caacac tctcaatc SAC002M24
BARCODE caacac gagtgg DS027M14
BARCODE caacac ccacgtc DS021M15
BARCODE caacac ttctcagc DS015M16
BARCODE caacac ctagg DS016M17
BARCODE caacac tgctta DS040M18
BARCODE caacac gcgaagt DS022M19
BARCODE caacac aatcctat NC027M20
BARCODE caacac atctg NC028M21
BARCODE atcggtt aggaa SAC003M25
BARCODE atcggtt gagtgg SAC005M26
############################################
I ran make.contigs from mothur v.1.39.1 like the following:
mothur > make.contigs(file=flea.txt, oligos=oligos-map4mothur.tab, bdiffs=1, pdiffs=2, checkorient=t, processors=7)
It ran without errors. However, by what was printed on the screen, I am afraid it is considering the whole fastq content as my samples, instead of only the sequences containing the barcodes depicted in the oligo file for each sample.
############################################
It took 2327 secs to process 5327581 sequences.
Group count:
BfleaM1 120317
DS006M12 334566
DS015M16 10615
DS016M17 19446
DS021M15 9775
DS022M19 13734
DS024M8 295246
DS027M14 20810
DS040M18 7933
FOF007M7 321275
FOF012M11 261574
FfleaM3 204135
NC014M10 257191
NC027M20 14456
NC028M21 14559
NC036M22 10184
NOfleaM5 304042
SAC001M23 10311
SAC002M24 12102
SAC003M25 26277
SAC004M9 242164
SAC005M26 21569
SAC015M13 12304
SAC023M6 328508
SfleaM4 308685
XfleaM2 241776
Total of all groups is 3423554
Output File Names:
flea.trim.contigs.fasta
flea.trim.contigs.qual
flea.contigs.report
flea.scrap.contigs.fasta
flea.scrap.contigs.qual
flea.contigs.groups
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.
####################################################
Can anyone shed a light on how mothur would consider only the sequences with the target-barcodes (on the oligo file) within each fastq pair?
Using 7 processors.
x�vyK�b���٥sӪ��’%AR1��n�LI����bØѶ is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
�l*���+=������V��u���g�55S�6v^r�ui�����C������csS%��!�i0�?�ttQTQ��j���1�"�S"!�<�J�8�5G�B$�X<'R��h is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
p4�����"� is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
�Yʖ͖f�r�덼�9x��I��$�G’m�t&���~ is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
�-2�J���i-$ is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
��’ is in your forward fastq file and not in your reverse file, please remove it using the remove.seqs command before proceeding.
##########################################################
Here’s the oligo file for the M1 sample only:
$ cat oligos-map4mothur-M1.tab
primer CAGCMGCCGCGGTAATWC CCGTCAATTCCTTTRAGGTT 519F926R
BARCODE ggtac aggaa BfleaM1
BARCODE tgattgac tctcaatc Blank
BARCODE ggtac tctcaatc D1
BARCODE caacac tctcaatc D13
BARCODE atcggtt tctcaatc D25
Looking forward to hear from you,
Thanks very much,
Elton
Can you repost this on a new thread? This thread is quite old at this point.
Pat