# make.contigs and demultiplexing **URL:** \ **Category:** Commands in mothur\ **Created:** [March 31, 2015, 12:04pm UTC](https://forum.mothur.org/t/make-contigs-and-demultiplexing/2335 "2015-03-31T12:04:57Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![ameziti](https://avatars.discourse-cdn.com/v4/letter/a/73ab20/32.png) [@ameziti](https://forum.mothur.org/u/ameziti)\ **Post date:** [March 31, 2015, 12:04pm UTC](https://forum.mothur.org/t/make-contigs-and-demultiplexing/2335/1 "2015-03-31T12:04:57Z")
Hello, I have a problem with make.contigs and demultiplexing. I have 2 .fastq files (one forward and one reverse) containing seqs from 40 samples. After [mothur \> make.contigs(file=stability.files, processors=8)] the assembly is fine but some of them are reverse complements. I tried different versions of make contigs such as [mothur \> make.contigs(ffastq=./SAMP1-40\_S2\_L001\_R1\_001.fastq, rfastq=./SAMP1-40\_S2\_L001\_R2\_001.fastq,oligos=./test.oligos, checkorient=t)] but again it does not seem to work since all sequences go to ‘./SAMP1-40\_S2\_L001\_R1\_001.scrap.contigs.fasta’. Do you know how i could set the orientation of my contigs to always start from my barcodes and my forward primer? ---
**Author:** ![westcott](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/westcott/32/18_2.png) [@westcott](https://forum.mothur.org/u/westcott)\ **Post date:** [April 1, 2015, 1:48pm UTC](https://forum.mothur.org/t/make-contigs-and-demultiplexing/2335/2 "2015-04-01T13:48:23Z")
Could you post your oligos file and a few sequence from your forward and reverse files? ---
**Author:** ![ameziti](https://avatars.discourse-cdn.com/v4/letter/a/73ab20/32.png) [@ameziti](https://forum.mothur.org/u/ameziti)\ **Post date:** [April 1, 2015, 6:45pm UTC](https://forum.mothur.org/t/make-contigs-and-demultiplexing/2335/3 "2015-04-01T18:45:22Z")
here you can see two seqs @M02542:38:000000000-ABF47:1:1101:19393:1063 1:N:0:2 ACATAAAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAANAAGCNNNNAGTNNGACTTTGGGTTGGGTCGCCCGGTCCGCCTTTGGTGTGNACCGNGCNGCTCNTCNCTAANGCCGGCGATGCGCTCCTGTCCTNAACTGGCNGGGTCGTGCCTCCGGTGCTGTNACTTTGAAGNAATTAGAGNGNTCAAAGCAAGNCNACGCTCNGNATACATTAGCNTNGGATAACATCATAGGATTTCGGNCCTATTACGTTGNCNTNCGGGATCGGAGTAATGNTT + CCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG#:DFG####::D##:CFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGD#:BFG#:B#:BFF#:B#:B@F#:BFGGGGGGGGGGGGGGGGGGG#6@FGGGG#5@FGGGGGGGFGGGGGGGGGG#4=CFGGGGC#3=FEFGGF#3#3=CFDGGGG8#2#/2\>EC8#\*#\*2:FFGGGGF#2#2:CFGFGGGGFFGFGFFGGFG\>#.1\>DFFFF\>FF5#,#.#(.8?FE@GFBFFFFA)#-4 @M02542:38:000000000-ABF47:1:1101:12170:1063 1:N:0:2 GAACCCAAACACTTTGGTTTCCCGTAAGGCGCCGAATGAGTCATTAAAAATTAACATCATNCGATNNNNAGTNNGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCNTCTTNGANCCCCNAANTTTCNTTCTTGATTAATGAAAACATCCNTGGCAAANTCTTTCGCAGTAGTTAGTCTTNAATAAATCCNAGAATTTCACNTCTGACAATTNANTACTAATGNCCCCAACTATNCNTATTAATCATTACGTCGATCTTAGAAACCAACAAANTNANATCAAACGTCCTATTTNAT + CCCCCGGGCFGGGFGGGFGGGGGGGGEFGGGGGGGGGGGDEFFGGGFFGGGGFGGGGCFG#:CF@####96:##:::CCCFGGGGGGFFGGEFGGGGDGGGGGGGFEFGGG#+:DF#::#:BFG#:B#:BDD#:BFFG9FGGGDFFFGGGGFGGG#8\D@FECFGG#16DFFGGGGC#6#\*49=\FFFCDFG#(#0#–83\<4?E\>F??BFFB#(- ################# And here is the .oligos primer CAGCCGCGGTAATTCCAGCT CAGCCGCGGTAATTCCAGCT BARCODE ACACTGTC ACACTGTC D2.1.ns31 BARCODE ACACTTGC ACACTTGC D2.2.ns31 BARCODE ACAGAAAA ACAGAAAA D2.3.ns31 BARCODE ACAGAGAG ACAGAGAG D2.4.ns31 BARCODE ACAGATTG ACAGATTG D2.5.ns31 BARCODE ACAGCAAA ACAGCAAA D2.6.ns31 BARCODE ACAGCGAG ACAGCGAG D2.7.ns31 BARCODE ACAGCGGC ACAGCGGC D2.8.ns31 BARCODE ACAGCTTG ACAGCTTG D2.9.ns31 BARCODE ACAGGCCA ACAGGCCA D2.10.ns31 BARCODE ACAGTCTT ACAGTCTT D2.11.ns31 BARCODE ACAGTGCG ACAGTGCG D2.12.ns31 BARCODE ACATAAAG ACATAAAG D2.13.ns31 BARCODE ACATCACC ACATCACC D2.14.ns31 BARCODE ACATCCCG ACATCCCG D2.15.ns31 # In total we have 40 samples but only 2 fastq files # As you will see the first sequence corresponds to sample D2.13.ns31 but the second sequence starts from the reverse primer. # As a result half of my contigs start with the reverse primer(no barcode) and I cannot demultiplex Thanks a lot!