Hi all.
Sorry to be a bother but I just did my first ever miseq run for 16S rRNA sequencing. I got four files back the paired end reads and two index files. I used the nextera XT index kit for dual barcodes
If I run make.contigs with the four column file option I get no contigs, everything goes to scrap
If I run make contigs without the barcode files I get contigs
The barcode files have sequences that match the 5’-3’ direction in which they were designed.\ (both forward and reverse)
I have tried with an oligo file that includes both the 5’-3’ barcodes and the reverse complement of the reverse barcode, still everything goes to the scap.contigs.
Any ideas/suggestions?
Thanks
Could you post a few reads and the barcodes?
Here you go…
Forward reads
@MISEQ:174:000000000-ABRWE:1:1101:15774:1378 1:N:0:
TTGCAGCAGCCGCGGTAAGACGAAGGTGGCGAGCGTTACTCGGAATTACTAGGCGTAAAGCGCGAGCAGGCGGTTCGGCAAGTCTGTTGTGGAAGCTCCGGGCTCAACCCGGAGAGGCCAATGGATACTGCCGGACTTGAATACGGGAGAGGTCGCTGGAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAGGAACACCGAAGGCGAAAGCAGGTGACTGGACCGTTATTGACGCTGAGTCGCGA
+
1>11111>11>AAAEE?011BEE0EGFGGHGCGAEGGCFHHGG?FFGFEHF@FFEG/E/FEFCGGGC?FHEEEGGE@CG@@FHFFDGGHHCGGDG<F1?AC?-C.CFGFGGG?C-.?ACGEG/G:G0;;00CA?-@AFF09;9C.EG??.CBB9@@FF@-/;F9BFFFFFFFFB@@A@9BF;FBBF-@9;/B9/99/AB;-ABFB;>9@F?@-@@?FFFFABBFFFA/AF?FF-AFF//A?;-;AFB;;@#
@MISEQ:174:000000000-ABRWE:1:1101:15116:1382 1:N:0:
TTGCCAGCAGCCGCGGTAATACCAGCTCTTCGAGTGGTATTCATGTTTATTGGGCTTAAAGCGTTCGTAGCCTGTTTAGTAAGTCCCTTCTTAAATCCTGTCGCTTAACCTTTGGCCTGCTGTTGATACTGCTTTGCTTGGGGGCGGGAGAGGGTAACGGTATTCCATGGGTAGGGGTGAAATCTTATGATCTCTGGAGGACCACCAGTGGCGAAGGCGGTTGCCTAGAACGTGCCCGACGGTGAGGGACG
+
AA11>B1>B1>A1EA0EA0EFG1B1AFBHFEAAFFGAFA222DA22DFDBGF1FGGH1DD1BAF?FEGFFEG1GEGH21BB2BGH111B0BF@B@B2B1111/??F?>111B1101BBC01111BBB2B>BF1111BB1FE////AC–A…:…//0.AAEAC0;000;CCB…;BFGEFF;00//;9:B;B/;/BF?F?AAFFEFFFBFE@-9;BA=@@@@?FF/BFFFB-9A-9-=@@9BA-FFFFE@
@MISEQ:174:000000000-ABRWE:1:1101:14920:1392 1:N:0:
TTTCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGTTTGGTCAGTCGGATGTGAAAGCCCCGGGCTTAACCTGGGAATTGCATTCGATACTGCCTGACTGGAGTGTGGTAGAGGGAAGCGGAATTCCGGGTGTAGCGGTGAAATGCGTAGAGATCCGGAGGAACATCAGTGGCGAAGGCGGCTTCCTGGGCCAATACTGACGCTGAGGTGCG
+
1>1>>3@B>1>A1EC0EA0BBABEDFGCGGGCDFEGGEB/DFEG/ABGDFGGBGGGE/BBFDE?EEEGE@EFGGGGGCEHF2FFHCCGC>FDDF?FC0<C@CG@CHF>FGHFHE0<FG0DDDGFF0D<@GHFHGHCFGFE/GHFCG?G0G:CFBBF?E@@GA;FBFBA@G@@FFF??F@-/:;;;ABF;EE/;BFF?A@??;AFF9;BFFFE@-=;BE@@@@@FFFBF?AAA-;;BFF:B-:B–:-B-9@
@MISEQ:174:000000000-ABRWE:1:1101:15141:1397 1:N:0:
TTGCCAGCCGCCGCGGTAATACGGAGGGAGCTAGCGTTGTTCGGAAATACTGGGCGTAAAGCGAACGTAGGCGGCTTTTCAAGTCAGGGGTGAAATCCCGGAGCTCAACTCCGGAACTGCCCTTGATACTGGATGGCTTGAATCCTGGTGAGGTCAGTGGAATTTCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGACTGACTGGACAGGTATTGACGCTGAGGTTCG
+
A11>1@>1A?A0AE0E00BBBGGACGG/EEFFHHEGEAFFHGG>//BDGHGGHEGG//>@1>?/>EHHHGGGG?CGGH22BFFG>GHF?CGEFG?<1</<–<F1FCG11<CC?C-<<<C.;C/00:CCCB/CB.FG.AAFC09CBF/;0;FBFBBFFFGF;F0;9.=FEFFF/;FFFBBF;FFEFFEEFBBB/9A-9A-FFFFBEFFBFF@@;-AE>-@@9/;BFF/9/;A/:;BF/;AABF-AEFFF@
@MISEQ:174:000000000-ABRWE:1:1101:16130:1400 1:N:0:
TTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATTCGGATTTACTGGGCGTAAAGCGCGTGTAGGCGGCTTTCTAAGTCGGATGTGAAAGCCCTGGGCTTAACCTGGGAATTGCATTCGATACTGCGTGGCTTGAGTCTGGTAGAGGGAAGTGGAATTCCTGGTGTAGCGGTGAAATTCGTAGATATCTGGAGGAACACCAGTGGCGAAGGCGACTTCCTGGGTCAAGACTGACGCTGAGGTTCG
Reverse reads
@MISEQ:174:000000000-ABRWE:1:1101:15774:1378 4:N:0:
TGACTACAGGGGTTTCTACTCCTGTTTGCTCCCCCGGCTTTCGCGACTCAGCGTCAATAACGGTCCAGTCACCTGCTTTCGCCTTCGGTGTTCCTCCTGATATCTACGCATTTCACCGCTACACCAGGAATTCCAGCGACCTCTCCCGTATTCAAGTCCGGCAGTATCCATTGGCCTCTCCGGGTTGAGCCCGGTGCTTCCACAACAGACTTGCCGAACCGCCTGCTCGCGCTTTACGCCTAGTAATTCCG
+
111>>B111111AAEDGB3BBCG1GGH0FG1C0AA0/AEEHH//A/AEA11E/EEEEF2GFGGGE@@GHHFHGGFGGHHHECEEGHHCGFEFHGHGHHH1FBFFHBDFC/CCHG2D-@>@BAFF/A=-@@BE################
@MISEQ:174:000000000-ABRWE:1:1101:15116:1382 4:N:0:
TTACTACTCGGGTTTCTAATCCGTTTTGCTCCCCTCGCTTTCGTCCCTCACCGTCGGGCGCGTTCTCGTCAACCGCCTTCGCCTCTGGTGTTCCTCCCTAGATCATTGGATTTCACCCTTACCCCTTGAATTCCGTTGCCCTCTCCCGTCTCCTAGCCTAGCAGTATCTCCTGCAGCTCGACGGTTAGGTCACTGGCTTTAACCTGTGACTTTCTAAACCGGCTACGTACGCTTTAAGCTCAGTAATTATG
+
111>>3@D1>11A1AAG33BBB0ABFA0BF1AAA0000BGFH?G?/AF00/0AFEA////>/EEFF2/?EE/@0EEEEEHEE//?F1FGE/?FFGF0/010<?11?2211?FGG2FDG/?1?00/?0011>1<1<>F..<FHCCC########################################################################################################
@MISEQ:174:000000000-ABRWE:1:1101:14920:1392 4:N:0:
TTACTACTAGGGTTTCTAATCCTGTTTGATCCCCACGCTTTCGCACCTCAGCGTCAGTATTGGCCCAGGAAGCCGCCTTCGCCACTGATGTTCCTCCGGATCTCTACGCATTTCACCGCTACACCCGGAATTCCGCTTCCCTCTACCACACTCCAGTCAGGCAGTATCGAATGCAATTCCCAGGTTAAGCCCGGGGCTTTCACATCCGACTGACCAAACCGCCTACGCGCGCTTTACGCCCAGTAATTCCG
+
111>>BDF3111BBEFGB3BBAFBGHH01D1A0A0EAEEFGHE?/AAFC1BCEEE/EHHHHA1FAAA/CAGFEGEGGGFHG?A>EFGBGGGHGFGFHCE?EEFBGB>E1ACB>BFGHGCC?@?FF<0ECGFCGGHH<>D>B133111B1BAF31BBBFAFGE00EAA0A0E0EEFGH//BAAFF11BAEEE/FAB1DGHFBBGFGHHHHGGFGFHA?/>/FGBGGGFHGH2BEAA?/BFGB1F//?FHF2B1GHG<>B<FFE//<?FFFFFG@<CGFFHF<D1</FFGH?<DDBCG--CC;0;:CHBCC-AEFGF09B.9EEBBCF;0:;0B?A@-:A;9B9FF?=BEE;B-9-9--;/:BFB##
@MISEQ:174:000000000-ABRWE:1:1101:16130:1400 4:N:0:
TGACTACCGGGGTTTCTACTCCTGTTTGCTCCCCACGCTTTCGCGTCTCAGCGTCAGTATTGGTCCAGGAAGTCGCCTTCGCCACTGGTGTTCCTCCAGATATCTACGCATTTCACCGCTACACCTGGAATTCCACTTCCCTCTATCTGACTCTAGTCGCGCAGTTTCGCATGCACTTCCCGGGTTGAGCCCCGGGCTTTCACATCCGACTTAGGAAGCCGCCTACTCTCGCTTTACGCCCAGTAATTCCG
Barcodes
BARCODE taaggcga ctctctat S1_L1_R1_V
BARCODE taaggcga tatcctct S1_L1_R2_V
BARCODE taaggcga agagtaga S1_L1_R3_V
BARCODE taaggcga gtaaggag S1_L2_R1_V
BARCODE taaggcga ctaagcct S1_L2_R2_V
BARCODE taaggcga gcgtaaga S1_L2_R3_V
BARCODE cgtactag ctctctat S1_L1_R1_D
BARCODE cgtactag tatcctct S1_L1_R2_D
BARCODE cgtactag agagtaga S1_L1_R3_D
BARCODE cgtactag gtaaggag S1_L2_R1_D
BARCODE cgtactag ctaagcct S1_L2_R2_D
BARCODE cgtactag gcgtaaga S1_L2_R3_D
BARCODE aggcagaa ctctctat S2_L1_R1_V
BARCODE aggcagaa tatcctct S2_L1_R2_V
BARCODE aggcagaa agagtaga S2_L1_R3_V
BARCODE aggcagaa gtaaggag S2_L2_R1_V
BARCODE aggcagaa ctaagcct S2_L2_R2_V
BARCODE aggcagaa gcgtaaga S2_L2_R3_V
BARCODE tcctgagc ctctctat S2_L1_R1_D
BARCODE tcctgagc tatcctct S2_L1_R2_D
BARCODE tcctgagc agagtaga S2_L1_R3_D
BARCODE tcctgagc gtaaggag S2_L2_R1_D
BARCODE tcctgagc ctaagcct S2_L2_R2_D
BARCODE tcctgagc gcgtaaga S2_L2_R3_D
BARCODE ggactcct ctctctat S3_L1_R1_V
BARCODE ggactcct tatcctct S3_L1_R2_V
BARCODE ggactcct agagtaga S3_L1_R3_V
BARCODE ggactcct gtaaggag S3_L2_R1_V
BARCODE ggactcct ctaagcct S3_L2_R2_V
BARCODE ggactcct gcgtaaga S3_L2_R3_V
BARCODE taggcatg ctctctat S3_L1_R1_D
BARCODE taggcatg tatcctct S3_L1_R2_D
BARCODE taggcatg agagtaga S3_L1_R3_D
BARCODE taggcatg gtaaggag S3_L2_R1_D
BARCODE taggcatg ctaagcct S3_L2_R2_D
BARCODE taggcatg gcgtaaga S3_L2_R3_D
Hope this helps. Thanks in advance for the help.