demultiplexing help- process_radtags, ddRADseq, single-end

[Joshua LaPergola]

Hi all! 

Any help with this would be greatly, greatly appreciated. 

I recently got the raw data (singled-end, NovaSeq 6000, 138 bp read-length) back for my ddRAD-seq library, prepared with mspI and ecoRI. I used two sets of barcodes, 8 index barcodes and 16 P1 barcodes, to create a library of 128 individuals. Unfortunately, the raw data did not come back demultiplexed to index groups and I'm struggling to get a reasonable result with process_radtags (STACKS 2.53). I tried demultiplexing with a barcode file containing both sets of barcodes but kept failing to get it to work. So, i'm trying now to demultiplex to just the index groups. Here is the code:

/home/jl101/software/stacks-2.53/bin/process_radtags -p /scratch/gpfs/jl101/PALMCHATS/ \
                   -b /scratch/gpfs/jl101/PALMCHATS/Palmchat_IndexOnlyBarcodes.txt \
                   -o /scratch/gpfs/jl101/PALMCHATS/Palmchat_IndexGroups_mspI_noc_drc \
                   -e mspI -r -q --inline_null --disable_rad_check --barcode_dist_1 1 \
                   -i gzfastq -y fastq

And here are the results: 

917924818 total sequences
784451930 barcode not found drops (85.5%)
    13118 low quality read drops (0.0%)
        0 RAD cutsite not found drops (0.0%)
133459770 retained reads (14.5%)

So, apparently the barcode is not being detected or is absent?? The weird thing, too, is that the resulting index groups are highly skewed, with most retained reads going to Index8 (highlighted in yellow below):

Barcode Filename Total NoRadTag LowQuality Retained ATCACG PALM_Idx1 41140 0 43 41097 CGATGT PALM_Idx2 212509 0 52 212457 TTAGGC PALM_Idx3 13633 0 48 13585 TGACCA PALM_Idx4 30022 0 72 29950 ACAGTG PALM_Idx5 49339 0 29 49310 GCCAAT PALM_Idx6 78246 0 46 78200 CAGATC PALM_Idx7 184736 0 45 184691 ACTTGA PALM_Idx8 132863263 0 12783 132850480

I've used this same protocol before but with the raw data already demultiplexed to index groups and did not have this problem. 

I've tried a few other tweaks to process_radtags but everything results in the same highly skewed lumping of reads into Index 8. 

Any advice/help would be greatly appreciated. 

Cheers,

Josh

[Catchen, Julian]

Hi Josh,

 

Can you post a few dozen lines from one of your raw sequencing files?

 

julian

[Joshua LaPergola]

Hi Julian,

Thanks for your response. Here are some of the lines from the raw data:

@A00741:264:HNMWJDRXY:1:2101:1054:1000 1:N:0:0 GNATGAATTCTGGCTGGGAAAGGGAGGCGCTGCCTCTTTTTCTGGATTCAAACCCTTGTTCTGTCAACAACGCGGAGACCAGACACAGGAAATTATCTGCCAGGTGACCCTGCAAAATAATGGTTTGGATGAGTTTGT + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1090:1000 1:N:0:0 ANTGGAATTCCCATTCCACTCGCAGCCCCTGTCTGCTTCCCATATATGTGAGGGCACTGATAGCATGCTGGAAAAAAGCGCCTTTTTTTGGGGTTGCTGGGGACGAATTATCAGATATGGGGAGGCTTCAGCTGGAGC + F#,,FF,FFFF:FFF:FFFFFFF:F,F,FF:FFF:,FF,,:FF:,FF:,:,,FF:F,::,F,F,F:,,FF:FFFF,:::,F::,F::FF:,FF:,F,FF:,F:,FFF:,F:,FF:F:F:,F,FF:F,F,FFF::FF,F @A00741:264:HNMWJDRXY:1:2101:1108:1000 1:N:0:0 CNTGGAATTCTTGCTCAGCTGCTTAATAGAGTGTGTCAGAGCTCTGCTGTCCTCTCTGGAAGTGGAACCTTCCCAGCACAACAGTTCTGCATCCAGTCCTGCCAACCTCAAATATCCCCCTGAGTCGTATCTTATACT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1127:1000 1:N:0:0 ANTGGAATTCTCAGTAGGTAATAATTGCTTGAGCAATCATCTGGGAATACCTGTGTTTGTATTACAGAGCTAACTTGCAAGAATACACTGGGAGTGGTGTACTTTTTGTGCTGAATTACTGGGATTTTCCAGGCATCT + F#FFFF:,F,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF: @A00741:264:HNMWJDRXY:1:2101:1181:1000 1:N:0:0 GNATGAATTCTGGGGACACTCTGAGTTCTTCTTGGGGTTTTGGGAGCCCGAGATCTCATCCAGAGTGTGTTTTTCTCCTCTCCATCCCTCCCTAACCCCAAGATGATCCTCTCCCAACCTCCCGTGCCAACCTGGTGC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF,FF,FFFFFFFFFFFFFFFFFFFFF,FF,FFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1199:1000 1:N:0:0 ANGAAATTCTCTCCAGCACATATGTATAATCCTTGTGGCTGTGTATGTCCTCTCCACACCTGTTCTCTGTGATGCATTTACTGCTGCTGGTACTGTAGAGACCAGGTTCAGCAGGTGTCAGAATATTCTAGGCTGAGT + F#:FFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF: @A00741:264:HNMWJDRXY:1:2101:1217:1000 1:N:0:0 GNATGAATTCTGCACTGCCACTACATTAACTGGACTTCACTTTGCTTCTGCACTCCAAGGATGAAAATTAAAGTGCTTTTAAAATAATTTTTAACTGGAGATTGAAGCTAGGTTGTTTTTTCATCCATCATTTTACGA + F#FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1235:1000 1:N:0:0 GNATGAATTCAGCTCTTTATTTGCAGGTGCCTAAATTTGTTGTAAATATGCATCTTTACATGAGATGCATTTTCAGGCTCCGTTTCTAGTTAATGAAATCATGCAACTTTATATAGTTACCTCAATATAGGCGTCTAA + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFF @A00741:264:HNMWJDRXY:1:2101:1253:1000 1:N:0:0 ANTGGAATTCAAAATCATTTTTGCAAGCTGGAGATACAGTCTGATAAAATATGGACAAATGCCTACAGATATGAATAAACACATGCATATATAAATTTGTGAGGAAAGCTCTTATAAAAATAAATGCCATACTGGGAG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1271:1000 1:N:0:0 GNTTGAATTCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGTCTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAGT + F#FFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1289:1000 1:N:0:0 GNTTGAATTCTTTGATTGTCCTTGCAAGCAAAAAGTGTAAAATTTGATATAAAGGGGAAAACAATTCATTATGTGTGGCTCAGTAATGAAGAAAGTGATCATACCATATTCAACTTCATGTGTATTTTTTTCTGCATC + F#FFF,,FFFFF:FFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFF:F:FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFF:FF,F:FFFFFFFFFFFF,FFFFFF @A00741:264:HNMWJDRXY:1:2101:1307:1000 1:N:0:0 ANTGGAATTCATTGGTATAAACAGTTCCATTTCCCCTGTGGCTAATATTTTGGGAGAGATAACATCTGAAAGAACTTCAGTCCTCACTGTGTTTTGTACTGGTGGTGTACAGTCATGCTATTTTGGCCTGCGATTCTT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1325:1000 1:N:0:0 CNTGGAATTCTGGGATTAGGGGGCAAAGAAACACACCAGGATGAGATCTTGGGGTCCCAAAACCCCATGGATGTTGTGCACGGGGTGAATTCGGGGTTCTGGTAGGGAAAATTCTGGGATCAGGGAGGGATGGAATCC + F#F:FFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,:FFFFFFFFFFFFFFFF::FF,,F,FF,FFFFFFFF,F::,FFFFFF,:,:FFFFF:FFFFF: @A00741:264:HNMWJDRXY:1:2101:1344:1000 1:N:0:0 GNCGTAATTCACAGACTAAATAAAAGAAAGAAGGTAAATTCCCTAAAAATAAAAACAATATTAATACCACATTTGCAAATGGAGGCACCTCAAACTGTTCATGTGACCACAAAGATGCTAAAACAAAGGGCTATTACA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1416:1000 1:N:0:0 GNTTGAATTCAACATGGTTTTTTTTGGCTTGGTAGAAACACTTCTAAGCAGAAAAATGGTGTCGAAGTTGTACAACCTCATGGTTGTGGTAAAATATTAGTATTAGAAATATACTTTTAATATAGAATAGTGATATAT + F#FFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FF:F @A00741:264:HNMWJDRXY:1:2101:1452:1000 1:N:0:0 GNTTGAATTCAAGGAATTTCTAAGTAGGCCTGTTCGCCTTCTGGAGGGATGACAAGACTAGAAGGATAATGTAGGTCTAATTTCCAGCTTAAATAGAAAAGAGGGAAAGAAGCAGGGTGCAAATTGTAAAAACCTGTC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF @A00741:264:HNMWJDRXY:1:2101:1488:1000 1:N:0:0 GNATGAATTCGATGATGCTTTGATCTTTCTCTGAATTTCAGCAATACTGTTTCGTGGATTTCTTTCTGACCCATTGCCTTATACTACAGCAAATTCTCAGTTTACACAACACTCATTACATCCACAAAGACATCAAAT + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:::FFFFFFFFFFFFFF:FFF:,F:FF:FFFF,FFFFFFFFFFFFFFF,FF:FF: @A00741:264:HNMWJDRXY:1:2101:1524:1000 1:N:0:0 GNATGAATTCTGAGCAGCTGATGACAGGAACACATTCCAACTCCTCAGGCCTGCTGACTGTTCCCACCACTTCCCAGCCTTCTGCAAGTGTAGCCTATAAAAGGGATAGGAATAAAGAATTGCCAAATTTTCAAGCTT + :#:FFFFFFFFFF:FF,,FF,F,F,,,FF::F:,:F,F:,,,,:,FFF,,,FF,:,F,F,FF,,F,:::,:,F,FF,F:FFFF:,:,,F,F,,F,,:,FF,,,F,F,,,F:FF,,,,F:,F:F,FFF:,::::F:,FF @A00741:264:HNMWJDRXY:1:2101:1561:1000 1:N:0:0 CNTGGAAATCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGCCTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAGT + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1579:1000 1:N:0:0 GNGATAATTCAGATGGAGACTCAGAAGGACCCTGTTTGATTGCATAAAAAGCTGACAAATTTATTGTTTTAGGAATAGTTCTCTCCATTCCTCGTACTCTCTACTCCTGGTAGCTTTCTAGCTTTTTTTATTTCTGCT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F:F:FFFFFF:FFFF:FFFF::FFFFFFFFFF:FFFFFFF,F::FF:FFFFFFF:FFFFFF,: @A00741:264:HNMWJDRXY:1:2101:1615:1000 1:N:0:0 GNTTGAATTCAGTTTGGGACCATGTCAATGACTTTGTATGTGCAGCTATGCACTGCCTCTTTGCTTCAAAGCCCTGCAGTGTGCTGATCACAGAGAAGCCATTACTTGACCTAACCTTGGTGCAACCATGTGTGGGGA + F#FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1633:1000 1:N:0:0 GNATGAATTCCAATTAGAATAAAGTGTTCCAGTCGGTATTAGCTTGAAAAATATCTGAATATGGAATAGCATTTTTTGAAGTTTTTTTTTAATTGTGTAGGTGGTTAAGTTGATAGGTAGGAGTGGGTGTTGTACAGG + F#FFFFFFFFFF,FFFFFFFFFFFFFFF:F:F:FFF::,FF::FFFF::F:F:,:FF,FFF,F:,:F,F:F:F:::F,,,:,,,F,F,F,,,,,F,,:,FF,,,:,,:,F,F,,,:,:,F,,F,FF,,:,,F,,,,F, @A00741:264:HNMWJDRXY:1:2101:1651:1000 1:N:0:0 GNATGAATTCAGGATGTGCTACATAAAATTCATGCTATGTTATGAACGTGATTTATCTGCTTCCTAGCATTTGAGAATTACGTGGCGAAGTTGCACTTAAAACAGGGGGAATCTTGCCACTATCTTCAACTGAGGAAT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFF,FFFFFFFFFFFFFFFFF,F @A00741:264:HNMWJDRXY:1:2101:1687:1000 1:N:0:0 CNGTCAATTCTTGCCTGATGAATCATAGAAGGGTTTAGGTTGAAAGGAACATCAAAGACCACCTCATTTCAATCCCCTGCCATGGGCAGTGATGCCTTCCACTGGAAGAGGTTGCTCAGAGTCCCATGAAGCCTGGTT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1705:1000 1:N:0:0 ANTGGAATTCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGCCTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAGT + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FF:FF:FFFFFF:F::FFFFFFFFF:FFF::FFFFFFFFFFF:FFF @A00741:264:HNMWJDRXY:1:2101:1723:1000 1:N:0:0 CNTGGAATTCCTGCTGAAACAATTTGGTAACACTATCCCCTTGCTAGGGAACAGTGTCTGTGGCATGGGAATGTCACAATGTCGTCGCCCCGTTTGTTTGTGTTAAAATAAAATTCTAACTCTCAAAGCCACCTCTGG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF::FFFFFFFFFFFFFFFFFF:F:FFFFFFF:FFFF::::FFF:FFFF:FFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1741:1000 1:N:0:0 CNTGGAATTCTAGTTTTGGTAAAAGGGTGTCTTTTTACTTGTACAGCCACTCTTTGCAATTGGGAAGCCTTTTTGTTTCACTCAGAGGTCTTAAATAAGGTTACTGTTATCCACTAATGTCATACTTAAGTTGTGGTC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1796:1000 1:N:0:0 ANCAAATTCTGTATTACTTTCCTTCCCCTATTTATTTGCTCAAATTTATTGATTTTGGATGCACACATTATCATATAATAATGATGACTGGGTTTGTCTTCAGTTAATTTAGTCAATTTGGAACTTAGTGGTGAGGTT + F#FFF:F:F:F::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFF:FFFFFFFFFF:FFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FF:FFFFFFF @A00741:264:HNMWJDRXY:1:2101:1814:1000 1:N:0:0 GNCGAAATTCCTTGGATATCAGGAATGGAATCATCTCGAGAGGCCACCAGGGCCGAAATTCTGGGATTAGGGGGCAAAGAAACCTCCCAGGATGAGATCTTGGGCTCCCAAAACCCCATGGACGTTGTGCACCAGGTG + F#FFFF::FFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:,FFF:,F @A00741:264:HNMWJDRXY:1:2101:1850:1000 1:N:0:0 GNCGTAATTCTGGGATTAGGGGGCAATGAAACCCCCCAGGATGAGATCTTGGGGTCCCAAAACCCCATGGATTTTGTGCACCGAGTGAATTCGGGGGTCTGGTAGGGAAAATTCTGGGATCAGGGAGGGATGGAATCC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:1868:1000 1:N:0:0 GNATGAATTCTATAGGCAGAGTGACACAGAGTTCCCTGGCAGGTGCAAGAGGGAAGTCTTTATTGCAAAACCAGGCCGTTTACGCAGTTCGGTCATTATCAGTTACATTCCATTTAAGCACAACAAATGTAATTCAAC + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFF:F:FFFFFFF:FFFFF,FF: @A00741:264:HNMWJDRXY:1:2101:1886:1000 1:N:0:0 ANTGGAATTCCTGTGTGAAGAGCAGACAGGGATATGTGGCATCGCTGAAACTGGAGCAGGACCAAGCTCCTCCTCAGCCCTTCCTTAAGGCTGCCCAGAAGTCACCTTCAGATGGAAAAGGTCAAGGATTCATCAAAG + F#F,:FF,FFF,::::FFFF::FF,F,FF:FFF:F::F:F,FF:,:,FF:,F:FFF,FFFFF:,,,,FFF::F,FF:FF,:FF,,FF,F:,F,F,FFFF:F,FFFFF:,:FFF:F:FFFF:,::F,F,FFFFFFF,F, @A00741:264:HNMWJDRXY:1:2101:1922:1000 1:N:0:0 ANTGGAATTCTGGGATTAGGGGGCAAAGAAACCCCCCAGGATGAGATCTTGGGGTCCCAAAACCCCATGGATGTTGTGCACGGGGTGAATTCGGGGGTCTGGTAGGGAAAATTCTGGGATCAGGGAGGGATGGAATCC + F#FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FF:FFFFFFF:FF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFF,FFF @A00741:264:HNMWJDRXY:1:2101:1976:1000 1:N:0:0 ANGGAAATTCTACTTAAGTTTCTGAAAGTAACTGAAAAAGCGGGAGTTTTCTTTTTCTTTTAAAGCCAAAGTCTTTATGTTTTCATACGAAGTTTCACAGGAAAATGGGCCAAAGAATACAATTAAATATGCAAATGG + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFF @A00741:264:HNMWJDRXY:1:2101:1994:1000 1:N:0:0 CNATCAATTCTCTTGTATTTTACTCTATTTTCATCTTCTCAGACCTGTATATGTGTTTGCACATGCCTTTAAGAAGGTCAGAACACAAGACTATAGGGTGTTTCATGTTAATAGATTAGCTGTTAAAGAGGAATAAAG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFF:FFFFFF:FFFFF @A00741:264:HNMWJDRXY:1:2101:2031:1000 1:N:0:0 GNTTGAATTCATATTATAAATTCCAAGTGTCCATCACACTAGCAGTCTACTTTTTGATACCCATCTGAGTTGATGCATCTGTTGCCAGCAGTACTGCTGGCAGTGGTGTAATTTGGGACCTTTTTTGTACCTTGGATC + F#FFF,:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2049:1000 1:N:0:0 GNCGTAATTCCTGAAGCACAGCCAGTACCTTGAACTCTACAGAATGAGACTGAAATAACGTAAAAGGTCAGGGAAGTTCAAAGACAGAGGTGCTGTTTTCCTAGCTGGGGACAGCACACTGTTCCCAGACCACGCTGG + F#FFF::,:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFF::F:FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2067:1000 1:N:0:0 GNATGAATTCCTTATGGAATTTATGGTTCTTCCCATTGTTCCTTTCATCTCTCAGTATCCAATTTTATTTATCAGCAGACCTACAGTTTCTTTGTAAAGACGAATCTCTCCTTCCATTCATCAATCAATGGAATCCTT + F#FFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFF,FFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2103:1000 1:N:0:0 GNATGAATTCAAATTGTGCCAAAGAGTTGAAAAAGAGGTGGGAAAAAAAGGAAAAATTAAGGTAAAAATATGCAGGAAAAGAAGGAGAAAATTAAAAAATTCAGGTGGTGTTTGGAGAGAAAAATTGGATGCGAAAAA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF,FF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F,F:FFFFFFF:FF,FFFFF:FFFF:F,F @A00741:264:HNMWJDRXY:1:2101:2139:1000 1:N:0:0 GNCGTAATTCTATTTTAAAAGTGCCCAAATTTTGGTCAGACAGCTGAATCTGCAGACATTACTCATGATCCACTGATCCACAGAAGAATTCTACCCTGATTTATTACACTTTGTTAAGAACACCGCCAGAGACTCTTC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2230:1000 1:N:0:0 ANGAAATTCTTGCATATAAAAAGCTCAATTATATATGATATCAACTTTTAATGCACAGCTCTTGGGGGAAAAAAAAAAAAAAAAAAAAAAAAGGTGGATTGTCTCAGGGTGGATTGAGTGGAATTTCATCCATAGCCC + F#FFF:F,F,FFFFFFFFFFF:FFFFFFFF:F,FFFFFF:FFFFFFF:FFFFFFFF:FFFFF:FFFFFFFFFFFFFFFF:FF:,FFF:FFF,,F,F,,:F:,,,,,,,,,,,,,,:,:,FF,,,F,,::F,,F,,,FF @A00741:264:HNMWJDRXY:1:2101:2248:1000 1:N:0:0 CNTGGAATTCAGACACATGACAGTTCCTCCTTACTGCCAGCCATCTGTGAGCCTCTAAAAGTGGCTCTGTTGTGAGAGCCTTGCTGGGTATCTTCTTCACCAGTTCTCCTTGAATAGTCTTCTCAAGCAAAGCAGTGA + F#FFFFF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FF,:FFFFFFF @A00741:264:HNMWJDRXY:1:2101:2302:1000 1:N:0:0 ANTGGAATTCTGGGGACACTCTGAGTTCTTCTTGGGGTTTTGGGAGCCCGAGATCTCATCCAGAGTGTGTTTTTCTCCTCTCCATCCCTCCCTAACCCCAAGATGATCCTCTCCCAACCTCCCGTGCCAACCTGGTGC + F#FFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFF,FFF,FF::FF,:FFFFFFF:FF:F:FF:F,FFF:FFF::FF:F:,FF,FF,FFFFF @A00741:264:HNMWJDRXY:1:2101:2356:1000 1:N:0:0 TNCATAATTCTTAGACAGAAGCACAGAATTAAACTGAACATACTGGCACAGCATCAACAAATCTTTCAGAGAAAGCCTGACAGTCCTGCACAATAAATCTGCTCCCTCTTCATTACAAGCCTGTCTGCAAGAACACTC + F#FFFF,FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2374:1000 1:N:0:0 CNTGGAATTCATAGTGGAAGTTCAGCATCTCTGGAAACTGCAGAGCCTCCTGCAGCTGAAACACCAGTTACTCGTAAGTTCAAGTCCACTGGAATTGGGCAAAACTCTTTGACCCTGCCAAATGAGTTAAATTAACAG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF @A00741:264:HNMWJDRXY:1:2101:2392:1000 1:N:0:0 GNATGAATTCCTACCCCAACATGGTGCACACATTCCTCAATTCTCCTTTTTAGGGCAAGATGGCAACACCTTTTCCACATCCTCTGTCAATAGTCCCTATCACTCCTCATCCATGCCATCTGAGCTCTGCGAATGGCA + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2428:1000 1:N:0:0 GNATGAATTCTATTCTCATTCCAAAGGTAATTTTCTCAGTATCCCTTAAGATTTAAAAATTGTTAAAAATTGTTCTGAGTTATAAACAAATACAGTCACTACTACAGTCAACAGTGCTACTACACAGACTTCATTTCT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:F @A00741:264:HNMWJDRXY:1:2101:2446:1000 1:N:0:0 ANTGGAATTCCAAACACATCTTCCTTGTATCTGAATGTTAATGCCCACAATTTCTTGTTCAACCCTCAAAGAGAGTATTGCAAGGCACACAACAAAAGGTTGGCATCACATCCTCTGTTCTTACCTTCCAATTCATGT + F#FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2465:1000 1:N:0:0 CNTGGAATTCAACTGCTCTAACTCGCTCTGACAACTACCATACGACTGAATCCTGAAAGGTCCCGTGTACCTGAGCATGTCAGCAGTAGATTTCTAGAACTGCAAAAGGTCAGCAGCACTGTGATTCACAGGACTTGT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2483:1000 1:N:0:0 CNTGGAATTCTCTTGAGATGAAGCTACGCATGATATCCAAGCAATTTTGGGGACACTCTGAGCTCTTCTTGGGGTTTTGGGACCCCAACGTCTCATCCAGAGTGCTTTTTCCCCATCTCCATCCCTGCCCTAGCCCAA + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FF:,FF,FFFF:FFFFFFFFFFFFFFFFFFFFFF: @A00741:264:HNMWJDRXY:1:2101:2501:1000 1:N:0:0 ANTACAATTCTAGTTTTAAGCCAGCTCTGTTTGAGAAAATTTGGGTTGAAATGTTTCCTAGTTATAACCACAGGGCCTCAAAACTTATGAAACAATTCCAATTGCACTGGTGAGAAGTCTTTATGCCTTTACATTGCT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2519:1000 1:N:0:0 GNCGTAATTCTTCTCACAGCAGTCACTTCTCAGTTCACTGCTCCCTTCATGTTGCCAGACTTTGGCATTTAACTTATGATGCATTCAGTGGAATTAACTCAAAGCCAGAGGTGAAGAAGAACCTGCAGGCAACTCTTC + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF @A00741:264:HNMWJDRXY:1:2101:2537:1000 1:N:0:0 GNTTGAATTCTTCAGTCACAATGCCCCTGACCCTTCCAGAAACTGCATTACAAGAAACAGAAGAAACCTCCCCTCAAGCAAGAATACTCTATAGTTACTGAAATTTACATCTTTGTTCTTTTCTGAACATTATCTTTC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2555:1000 1:N:0:0 ANTACAATTCTCTGCACTTGAGTGATCTTGAAGTCCACATATTTAACAGTTGTCTTTCCTATTTGAGTTTTTCTTTTAGTTACTCAGTAACCATTCTGTCCCAGAAAGTTCAACAGACTTATGATCATTTGTAGGCAT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFFFFFFF:FF @A00741:264:HNMWJDRXY:1:2101:2573:1000 1:N:0:0 CNTGGAATTCAACAATTAAGGATTAAGACAAAGTTTTTCATCCAACGGTAAAAATGGATTTTTGCTGCCGCTTAACATCAAAAACAAACCAAAACAAATTAGGTCAGGACACTAACATTAAGGATGTGGCTTCTTACA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2591:1000 1:N:0:0 GNTTGAATTCTCTTTGTTTCTTTCGCCACAGGTGTCAGCAGAACCTGGTCATCCCTTAGCGCGCAGATGTCCAGGAATTTCAAGTGAGCAGAAATGCAGTGTAAGGCCCTTCCAGAAGATGTTTTCTGCAGCCAGACA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2609:1000 1:N:0:0 GNTTGAATTCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGCCTTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2627:1000 1:N:0:0 GNGATAATTCCCTATATCCAAAGATTTCTGCCAACCAAAATCTGCCAGGGCTGACAAGTCTGGCTGGCCTTGGTTTTCTGAGGACTGGCACTCATCTGCCTTTGAAACACTGGGACTCTGTGCTTCCCTTCCTAATGA + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2682:1000 1:N:0:0 ANACAAATTCAGAAATTGCTATGACTCAATCCTTTAGTAAGATTTTGTTAGAACTGAAACTCTTCTTACTCATTCTACTTTCATTCTCACATCTACAGTGACTTCTAGGAATGGGCAGATCAAATTGCACATATTTAT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2700:1000 1:N:0:0 GNCGAAATTCTAATATTGTATATTAGAATTTTATATGTTTATATTATTAGATTATTATATTAGAATTTTATATGTTTATATTATAATATATAGAATTCTAGACTTGCATGGATTTCAGGGATTTTATTTACATGTGAG + F#FFF:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:F:FFFFF,FFF:FFFFFFFFFFFFFFFFF,FFFFF:F:F:F:,,,F:F,FF,:F:FFF,:FFF:,FFF,F,FF,FFF,:F,F,::FF,,FFFF:F @A00741:264:HNMWJDRXY:1:2101:2718:1000 1:N:0:0 GNATGAATTCTCAAATCACTAAATGTATGCGGACATCCTATGGATGTGGTGCACTGTCTAGGTCCAGGAGGTTGAGGGGGGCAGGGAAAATTCTAGGATTAGGGAGGGATGGAACCCCTTGAGAGGAGCTCAGAGCAT + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFF:FFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2754:1000 1:N:0:0 GNATGAATTCCACTGACAGAAGTCTGCCAGGTACTCAGAATCTCCTGAAACACTGAGTTCTAACTGCAGTCCTTTGTAAAACAGAACCCCTCTTATTCCTCCATCGTTCAACATACAAGTTCATTACAACAAGTTACT + :#FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF:FFF:FFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2808:1000 1:N:0:0 CNATCAATTCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGCCTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAGT + F#FFFFF,FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2844:1000 1:N:0:0 ANGGAAATTCCTTAAAATATGAGTTTAGGAAAACTGTTGGGAAAAAATCTGCATTTCCTAAAATTTACTGAAAAATCTTGGGAGGGAGGAGGGAGAAGGGGATGTTTGACTTTGTTTTTTTGTTATTTCCATTTTGTT + F#FFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:F:FFFFFF::FFFFFFFFFFFFF,FFF,F:F,:,,FFF,FFF:FF,FFF,F,,FFFF,:,,F,F,,,,FF,F,:,:,F,F,:,F::,FFF:,,: @A00741:264:HNMWJDRXY:1:2101:2862:1000 1:N:0:0 GNTTGAATTCTTTTCCACAGGAGAGGAAAATACCTCAAATCCCAGGAGCACTTACATAAAACACTGACACATGGATCTTGTAAACACTAAAGGTCAAACCAACTAAAGGTGCTGCTCTATAATGGTTCCATTTGGGTA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:F:FFFFFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFF:FFFFFFFFFF:FFFFFF:FF:FFF:FFFFFFFFFFFFFF: @A00741:264:HNMWJDRXY:1:2101:2880:1000 1:N:0:0 GNATGAATTCAATCAAAATTCCATAGATTAGGATGAAATGAGGCATGGGTCATTTACAGGGGAATTTAATTGCAGAGCCATTAGACAAATAGTATATTTGCCATTCTCACATTATCCATCATTTTAGACATCAATAGA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2899:1000 1:N:0:0 GNTTGAATTCTGTCCTCTGAAAGCAAAGTTCAGCTTAAAGCATTTTTAATTTGGTCCAAAAATATGATTAGCCTGGAATGTCTCCTTTTAAAAACAAAAAATCACATTATTCTTCAGTTAAATTTCTCATCCTTTATA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:2935:1000 1:N:0:0 CNTGGAATTCTCTTGAGATGAAGCTACGCATGATATCCAAGCAATTTTGGGGACACTCTGAGCTCTTCTTGGGGTTTTGGGACCCCAACGTCTCATCCAGAGTGCTTTTTCCCCATCTCCATCCCTGCCCTAGCCCAA + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FF,FFFFF:FFFFFFFFFFFFFFF:F @A00741:264:HNMWJDRXY:1:2101:2971:1000 1:N:0:0 GNTTGAATTCAGTTCTGCTGGGAAGCACAACTGGGCCTTCCTTCCTCATGAGGGTCCCTGGAAAGGTTCCTGTCTTGAAGTTCGTCTGTCTAGGTCCTTTCTGATTAAGTTCCTCTTAAGTCATGGCGCCATTACAGT + F#FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF::FFFFFFFFF,F,FFFFFFFFFFFFFFFFFFFFF:FFFFF: @A00741:264:HNMWJDRXY:1:2101:2989:1000 1:N:0:0 CNTGGAATTCCAGAGATCTCCGCCAGGGAGATCTCCCCACAGAGCTGTGGCTCTGTGGCCGCAAATCAAAGGCACGGAGCTGTGCCGAGGAATATTTTGCAAACATTTCTCTTTTTCAACCCAAAATGTTCTTGGTTG + F#FFFFF:FFFFFFFFFFFF:FFFFFFF::FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF,FFF,F:FFFFFFF,FFFFFFFF:FFFFFFFFFFFF,FF:FFFFFFF @A00741:264:HNMWJDRXY:1:2101:3007:1000 1:N:0:0 ANTGGAATTCCTTGGATATCAGGAATGGAATCATCTCGAGAAGCCACCAGGGCCGAAATTCTGGGATTAGGGGGCAAAGAAACCTCCCAGGATGAGATCTTGGGCTCCCAAAACCCCATGGACGTTGTGCACCAGGTG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3061:1000 1:N:0:0 GNTTGAATTCAATTATTCTTTGCCTATGATATTAGAAAAGGTACTGTGCTGTATGACTTTTCCACAAGGGCATTTTTAAAAACTGCATCTACATTTTATAGACTCACTTTGCATTGGAAGGGATTTCTGGAGGTCATC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3079:1000 1:N:0:0 GNATGAATTCAACATATTTACTTAAAAAATAAGATTAGAGGACACAAGATATATTGTTACTTTATAAAGGAATTTCTGAAATTAAAGAAATAGCCAAAAAAACCAGACATGATCTTAAGGTAGATTTTGGAACTAAAC + F#FF,FF:FF,F:FFF,FFFF::FFFF,FFFFFFFF,,:,FF,F,,F,F,F,F,FFFF:,F:F,F,F::FF,F,,:FFF:FFFFF,FFFFF,,:,,F,FFF,,FFF::,FFFF:,FF:,,,F,,,:FF,:,F,FF,,F @A00741:264:HNMWJDRXY:1:2101:3097:1000 1:N:0:0 GNATGAATTCAAACTATTTAACCATATATTTTAGCTTCAGAGTATTTTGCACTTTTAGCATCTTTTAAAACAGGCATAGCCCTGTGTCTGAGCTATTTTTTACTTACACCAGCAAATATGTTTTTGAAGAGAACACAA + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3134:1000 1:N:0:0 CNTGGAATTCACACAATAACAACAGAATATATGGTCCACCCATTTTTTCAGTTATATGTTTCAATAAAGAAACATGACCTGAATTTGGAGTCAGACTACAGATGAGAAAAACTGTCCGCACAGACATTTAATGAAGAT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3152:1000 1:N:0:0 GNATGAATTCTTTATTCCCCAAGGTAGATAGTTATTTCTAGGTTTATTATAATTGTTTCTCTATTTCTTCAAAAATAGTGCACTATACTCTACTCTACTATACTACTATATACTACTACTATATACGACTACTATATC + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3170:1000 1:N:0:0 ANTGGAATTCGTAGACTTTTCCCCTCAAAACTCCAACCCTCTCTCTCTTTTTTTTTTTTCTTCTTGTTCTGAGAGAGAGAGAGAAAGGGAGAGGGAGAGAAGTTGACTTGCGGCTGCATCTCAGCACGCACGTCAGCG + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F,FFF,F:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3188:1000 1:N:0:0 CNATCAATTCAAGGAACTGAAGAGGAGCTGTTGGTTTGATGGTTGGACTCTCAGAGGTCTTTGCCAACCAGAAGGATTGTGTGTGATCCTACGATTCTAAATGATTCCATGATCCCATGACTGAGCCTGCAGCACTTC + :#FFFFFFFFFFFFFFFFF::,::FFFFF,FF,FFF::FF:F:,F:F::FFFF:FF:F:FF,,:FFFFFFFFFF:FFFF,:F:::FFFF,F::F,:,F:F:F,FFF:,FFFFFF,FFFFFFFFFFFFF:FF,FF:,FF @A00741:264:HNMWJDRXY:1:2101:3206:1000 1:N:0:0 GNTTGAATTCACCCCATGCACAACATCCATGGGGTTTTGGGACCCCAAGATCTCATCCTGGGGGATTTCTTTGCCCCCTAATCCCAGTATTCCGCCCCTGGAAGCCTCTTGAGATGAACCTACGCACGATATCCAAGG + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3224:1000 1:N:0:0 GNATGAATTCAAATTATCAATTTGCATAAAACAGTGCTGTATTTATTAAAATATATATGCGTGTGTATATAATTAAGTGAGCCAATGCAATGCATGAGAAATGAATGGGAAGGTGGCCCATAATAAGGGTGTCAGTGA + F#FFF:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF,FFFFFFFFF,F:FFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3242:1000 1:N:0:0 CNATCAATTCAAGGAACTGAAGAGGAGCTGTTGGTTTGATGGTTGGACTCTCAGAGGTCTTTGCCAACCAGAAGGATTGTGTGTGATCCTACGATTCTAAATGATTCCATGATCCCATGACTGAGCCTGCAGCACTTC + F#FFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF,F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3296:1000 1:N:0:0 GNTTGAATTCTCCAAGTAAGTCAATCCACCCTGCCTCACATGAGATGCATACAGGGCTGTAGTCATCAAGTTACAGAAGTAAACTCAGAATTAAGGTTAAAATGTTTTTTTAAATTACCAATATTAATAATGTTGAGA + F#FFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3314:1000 1:N:0:0 GNCGTAATTCTTTTGTTGTTCTGCGTGATTTGCTACAGGTGCCATAAATATACTGGAATTCACGATCTGTGAAGTGCTGCCTCCAGAGCGGTGTGAGGAACAGCCCATAACCATAACAACCATAACACCCATAACCAA + F#FFF::,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3332:1000 1:N:0:0 GNATGAATTCTCTGTCTCACTGTGCAGGGCCAGGAGTTGGACTCAATGATCCTTGTGGTTCCCTTCCGACTCAGCATATGTTATGGTTCTGTGATTTTGTGATTGTAAAGGGAACTTTAGCACCTAGCTCACATGTAA + F#FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3351:1000 1:N:0:0 GNATGAATTCAGTTCTACTGGGCACCAGAACTCAACCTTCCTTCCTCGCAGGGATCCCTGGGAAGGCTCCCTTCTCGAAGTTCGTCTGTCTATGTCTTTTTTGATTAAGTTTCTCTGAAGACATGGCGATATTACAGT + F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3369:1000 1:N:0:0 ANACAAATTCATTGTAATATGTTTCCAACTTGAAAGAAATTTTCCAAAAAATTGTTTCAAGTTGCAAAATTCACTAAAATGAGTTTCTCTCTCTGTCTCTGCCTCTCTCTCTCTCTTTTTTTTTTTTTTTTTTTTTTT + F#FFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FFFFFFFFFF:FF:F:FFF,::F,:F:FFFFFFFFFFFFFFFFFFFF @A00741:264:HNMWJDRXY:1:2101:3387:1000 1:N:0:0 GNTTGAATTCTGGGATTAGGGGGCAAAGAAACCCCCCAGGATGAGATCTTGGGGTCCCAAAACCCCATGGATGTTGTGCACGGGGTGAATTCGGGGGTCTGGTAGGGAAAATTCTGGGATCAGGGAGGGATGGAATCC +

[Catchen, Julian]

Hi Josh,

 

Sorry, but this is not readable from here. Please post it in a form that preserves the lines.

.

[Joshua LaPergola]

How's this?

[Catchen, Julian]

I am going to reply here, so your original message is quoted below.

 

So, there are no index barcodes in the sequences you posted, so I would suggest you contact your sequence provider to understand what happened.

 

For the remainder of your message, you discuss trying (and mostly failing) to demultiplex “to just the index groups”, but this, by definition will not work. The index barcodes would have been encoded in the FASTA header line. If you are going to try any demultiplexing with these data, you will have to use the inline, embedded barcodes. But, I would try to understand what your sequencing provider did, first.

 

Best,

julian

 

 

From: stacks...@googlegroups.com <stacks...@googlegroups.com> on behalf of Joshua LaPergola <jlap...@gmail.com>

Date: Friday, December 17, 2021 at 10:24 AM
To: Stacks <stacks...@googlegroups.com>