I’ve been having trouble processing one set of sequence data (Titanium GS flex +). After extracting using sffinfo, I’m running trim.flows(flow=iron.flow, oligos=iron.oligos, order=B, pdiffs=5, bdiffs=2, maxhomop=8, minflows=200, maxflows=1050, fasta=T). (I’ve tried various minflows and maxflows values). Everything seems to go into scrap.flows. I’ve opened scrap.flows and sequences are thrown out at the barcode and primer level. I’ve opened up the fasta file and checked the barcode and primer in the oligos file, but can’t seem to figure out why they’re being thrown out.
Thanks!
Can you post the primers/barcdoes you are using along with ~10 fasta sequences?
Once we get this figured out, you’re going to need to use minflows and maxflows values that are the same.
Pat
Thanks Pat!
I’m using 27Fmod (5’-AGRGTTTGATCMTGGCTCAG-3’) and reverse 519 (5’-TTACCGCGGCTGCTGGCAC-3’)
Here is the oligos file
#forward AGRGTTTGATCMTGGCTCAG
forward AGGGTTTGATCATGGCTCAG
barcode AATAGCGG Green
barcode AATACGCC Mixed
and a few fasta sequences
IH7IBDS01C3JPF xy=1153_1777
AATAGCGGAGTAGTTTGATCATGGCTCAGATTGACGCTGGCGCAGGCTTAACACATGCAAGTCGAGCGGAAACGNTGGTANCTTGCTACCAGGCGTCGAGCGGCGGACGGTNAGTATGCTTAGGATCTGCCTAGTAGTGGGGATACGGCCCGGGAACTCGGATTAANACCGCATACGCCGTACGGGGAAGCAGGGATCTTCGACCTGCGCTATTAGATGAGCCTAAGTCGGATAGCTTGTTGGTGGGTAATGCCTACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCA
IH7IBDS01C4Q5G xy=1167_738
AATACGCCAGGGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAAACGATGGTAGCTTGCTACCAGGCGTCGAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCCAGTAGTGGGGAGTAGCCCGGGGAACGTACGGATTAATACCGCATACGCCCTTTTCGGGGTAAAGGACAGGGGAGTCTTCGGACCTTGCGCTATTGGATGAGCCTAAGTCGGATTAGCTTGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGGCAGCAGTGGGGAAGTCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTAGGGTCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
IH7IBDS01C7XH6 xy=1203_1484
AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGAGATCTTCGGATCTAGCGGCGGACGGGTGAGTAACGCGTGGGAATATGCCCTTCTCTACGGAATAGCCTCGGGAAACTGGGAGTAATACCGTATACGCCTCGGGAAGGATTATCGAGAGATAGCCGCGTGATAGTAGTGTGGGTAGTGCTACCAGCGACGATCATAGCTGGTTTGTAGAGGATGATCAGCCACACTGGGACGTGAGACACGGCCCACGACTCCTACGGGAGGCGAGCAGTGGGGAAGTCTTAGACAATAGGGGGAAACCCGTAGATCTAGCCATGCCGCGTGAGCGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCAGCTGGGAAGATAATGACGGTACCAGCAGAAGAAGCCCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAAT
IH7IBDS01D7NBE xy=1610_824
AATAGCGGAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAACACATGCAAGTCGAGGGGTAACAGGAGAGCTTGCTCTTGCTGACGACCGCGCACGGGTGCGTAACGCGTATACAATCTACCTATTACTAAGAGATAGCCCACGAGAAATTTAGGTATTAATAATTTTATTGGTATTACAATTTGGTCATCAAGTTAGTAATTAAAGGTTACGGTAATAGATGAGTATGCGTCCTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCTACGATAGGTAGGGGTCCGTGAGAGGGAGATCCCCCACCACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGACAATGGGAGCAATCCTGATCCAGCCATGCCGCGTGCAGGAAGACTGCCCTATGGGTTGTAAACTGCTTTTATACAGGAAGAAAAAGGTGTACGTGTACACTGTTGACGGTACTGTAAGAATAAGGATCGGCTAACTCCGTGCCAG
IH7IBDS01AN2V5 xy=157_531
AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGCGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGAAAGATTTATCGGTAAGGATTGGCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTTAGGGTCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACT
IH7IBDS01CNS9B xy=974_845
AATACGCCAGAGTTTGATCATGGCTCAGAATGAACGCTGGCGGTAGGCTTAACACATGCAAGTCGGACGGAAACGATAGAGAAGCTTGCTTTTCTAGGCGTCGAGTGGCGGACGGGTGAGTAACGCGTGGGAATCTACCCTATAGTTGGGGACAACGTATGGAAACGTACGCTAATACCGAATATGCTCTACGGAGTAAAGGAGCCCTCTTCCTGAAAGGTTTCGCTATAGGATGAGCCCGCGTGAGATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATCTCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGACGAAAGTCTGATGCAGCCATACCGCGTGTGTGAAGAAGGCCCGAGGGTTGTAAAGCACTTTCAATTGTGAGGAAGGTAGTGTAGTTAATACCTGCATTGCTTGACGTTAACTTTAGAAGAAGCACCGGCTAACTCTGTGCC
IH7IBDS01DS6QB xy=1445_1985
AATACGCCAGGGTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGGCGGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGGAAAGGATTTATCGGTAAGGATTGGCCCGCGTAAGATTAGATAATTGGTGGGGTAAGTGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGTAGGATGATCAGCAACACTGGGGACTGAGACACGGCCCAGACTCCTACGGGAGGGCAGCAGTGGGGAAGTCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTAGGGTCGTAAAGCACTTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
IH7IBDS01CGMZ0 xy=892_2014
AATACGCCAGAGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCTTCGGGTGAGCGGCGGACGGGTTAGTAACGCGTGGGAACGTACCCTTTTCTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATAAGCCCTTCGGGGGAAAGATTTATCGGGAAAGGATCGGCCCGCGTTAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTATAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGTCTTAGGATCGTAAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCCGCCGCGGTAATT
IH7IBDS01CNVY2 xy=975_268
AATAGCGGAGGGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCCTAACACATGCAAGTCGAGGGGTAACAGGAGAGCTTGCTCTTGCTGACGACCGGCGCGACGGGTGCGTAACGCGTATACAATCTACCTATTACTAAGAGATACGCCCAGAGTAAATTTGGATTAATATTTTATGGTATTACAATTTGGCATCAAGTTGTAAATTAAAGGTTACGGTAATAGATGAGTATGCGTCCTATTAGCTAGATGGTAAGGTAACGGCTTACCATGGCTACGATAGGGTAGGGGTCCTGA
IH7IBDS01DE09S xy=1284_1182
AATACGCCAGGGTTTGATCATGGCTCAGAACGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGCACCCTTCGGGGCGAGCGGCGGACGGGTTAGTAACGCGTGGGAACATACCCTTTACTACGGAATAGCCTCGGGAAACTGAGAGTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGTAAAGGATTGGCCCGCGTAAGATTAGATAGTTGGTGGGGTAATGGCCTACCAAGTCTACGATCTTTAGCTGGTTTTAGAGGATGATCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGAATGAAGGGCCTTTAGGGTCGTAAAAGCACTTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCAGCGCGGTAATT
A couple things…
First, you want this…
forward AGRGTTTGATCMTGGCTCAG
#forward AGGGTTTGATCATGGCTCAG
barcode AATAGCGG Green
barcode AATACGCC Mixed
Second, are you positive that you have flow order B? What happens if you try A with minflows/maxflows=450? The reads you posted aren’t super long (~550 nt long?), which makes me think you likely have A.
Third, if you are running mothur on mac/linux can you send the output of running this on your scrap.flow file?
cut -f 1 -d " " *scrap.flow | cut -f 2 -d "|" | sort | uniq -c
Pat
Thanks Pat
Changing the order seemed to do the trick… I was told the sequencing was done with the new chemistry on a GS flx + platform, but this indeed may not be the case…