Good Morning,
New to mothur, I have processed this data with qiime but ended up greater than 50% unidentified sequences, hoping that the ITS database in mothur helps identify more sequences… I attempted to demultiplex my fastq data using the make.contigs command but have been unsuccessful. The command I ran is : mothur > make.contigs(file=stability.files, oligos=barcodes.oligos, processors=1), and output:
…
7896229
Done.
It took 21989 secs to process 14625278 sequences.
Output File Names: stability.trim.contigs.fasta stability.contigs.report stability.scrap.contigs.fasta stability.contigs.groups
[WARNING]: your sequence names contained ‘:’. I changed them to ‘_’ to avoid problems in your downstream analysis.
mothur > summar.seqs(fasta=stability.trim.contigs.fasta)
mothur > summary.seqs(fasta=stability.trim.contigs.fasta)
[ERROR]: stability.trim.contigs.fasta is blank, aborting.
Using stability.trim.contigs.fasta as input file for the fasta parameter.
Using 1 processors.
[ERROR]: stability.trim.contigs.fasta is blank. Please correct.
Error in reading your fastafile, at position -1. Blank name.
I received two pairs of fastq files from the sequencing facililty (forward and reverse reads). They split our samples (n=144) into two runs, so the first pair of fastq files is for samples 1-72 and the second is for 73-144. I made an oligos file with the fwd and rev primers on the first line, and then the fwd and rev barcodes after that. I put several columns of descriptors following the barcodes for analysis purposes later… This is how we do it qiime so not sure if this was correct here as well. The facility only gave me fwd barcodes so I used an online tool to get the reverse barcodes, but had to do every single one manually…not sure if thats where I could have gone wrong.
This is what my oligos file looks like:
primer CTTGGTCATTTAGAGGAAGTAA TCCTCCGCTTATTGATATGC ITS
BARCODE AGATTTAG CTAAATCT 13.AXIL Allergic Axilla 13
BARCODE AGCAAGAC GTCTTGCT 13.EAR Allergic Ear 13
BARCODE AGCATCTT AAGATGCT 13.GROI Allergic Groin 13
BARCODE AGCAAATG CATTTGCT 13.ITDI Allergic Interdigital 13
BARCODE AGATTACG CGTAATCT 13.LUMB Allergic Lumbar 13
BARCODE AGCACCAA TTGGTGCT 13.NOST Allergic Nostril 13
BARCODE AGCCATAG CTATGGCT 14.AXIL Allergic Axilla 14
BARCODE AGCCCTCG CGAGGGCT 14.EAR Allergic Ear 14
BARCODE AGCGCACT AGTGCGCT 14.GROI Allergic Groin 14
BARCODE AGCCCGCG CGCGGGCT 14.ITDI Allergic Interdigital 14
BARCODE AGCATTCA TGAATGCT 14.LUMB Allergic Lumbar 14
BARCODE AGCGACAG CTGTCGCT 14.NOST Allergic Nostril 14
stability file:
Sam1-72_S1_L001_R1_001.fastq Sam1-72_S1_L001_R2_001.fastq
Sam73-144_S2_L001_R1_001.fastq Sam73-144_S2_L001_R2_001.fastq
Everything got thrown into the scraps file…
mothur > summary.seqs(fasta=stability.scrap.contigs.fasta)
Using 1 processors.
Start End NBases Ambigs Polymer NumSeqs
Minimum: 1 23 23 0 2 1
2.5%-tile: 1 371 371 0 4 365632
25%-tile: 1 575 575 0 5 3656320
Median: 1 580 580 1 6 7312640
75%-tile: 1 590 590 3 7 10968959
97.5%-tile: 1 599 599 11 8 14259647
Maximum: 1 603 602 84 301 14625278
Mean: 1 569.715 569.712 2.37107 6.08527
of Seqs: 14625278
Output File Names:
stability.scrap.contigs.summary
Could someone help me to understand where I went wrong?
Thank you,
Courtney