Demultiplexing failures in MiSeq run
Hi All!
I am sequencing (on a MySeq platform) a custom library containing 4 i7 indexes. In principle, it works, but when I look on demultiplexing stats, there is quite a large proportion of unrecognized indexes (resulting in about 20% of all reads unassigned). My adapter primers that I used to amplify the library were PAGE-purified. I pasted the detailed stats below. What do you think might be the reason?
Cheers,
Lech
I am sequencing (on a MySeq platform) a custom library containing 4 i7 indexes. In principle, it works, but when I look on demultiplexing stats, there is quite a large proportion of unrecognized indexes (resulting in about 20% of all reads unassigned). My adapter primers that I used to amplify the library were PAGE-purified. I pasted the detailed stats below. What do you think might be the reason?
Cheers,
Lech
Code:
SampleNumber 0 1 2 3 4 SampleName None 1 2 3 4 s_1_1101 12.85159 14.97901 24.74675 29.02787 18.39477 s_1_1102 12.86999 15.04931 24.61071 29.25371 18.21628 ### Most Popular Index Sequences ### Columns: Sequence ReverseComplement HitCount Index ACATTA TAATGT 209049 TGCACG CGTGCA 178081 GATCAC GTGATC 118236 CAGCGT ACGCTG 108928 GCTTAC GTAAGC 4512 GATAAC GTTATC 4442 ACATAA TTATGT 4079 GCTCAC GTGAGC 3643 GATTAC GTAATC 2996 GTTCAC GTGAAC 2485 GTTTAC GTAAAC 2366 GGTAAC GTTACC 2306 ACATTC GAATGT 1843 GTTAAC GTTAAC 1577 TGCAAG CTTGCA 1403 GCTTTC GAAAGC 1395 GTTGAC GTCAAC 1336 TGCACC GGTGCA 1303 GTTTAT ATAAAC 1246 GTTCAT ATGAAC 1241 GGTTAC GTAACC 1215 CATCGT ACGATG 1166 GCTAAC GTTAGC 1104 GGTCAC GTGACC 1066 ACTTTC GAAAGT 977 GGTGAC GTCACC 933 CATCAT ATGATG 922 CCTTTC GAAAGG 877 ACTTTA TAAAGT 848 TGCACA TGTGCA 815 CAGCAT ATGCTG 812 GATGAC GTCATC 794 ACTTAA TTAAGT 768 TTCCCT AGGGAA 726 GCTTAA TTAAGC 707 GTTTAA TTAAAC 684 ACTTAC GTAAGT 675 CCCTTC GAAGGG 675 TGTAAC GTTACA 671 GATCAT ATGATC 657 CCTCAC GTGAGG 642 TGCACT AGTGCA 639 GTTGAT ATCAAC 639 CCTTAC GTAAGG 633 ACATAC GTATGT 624 TGTACG CGTACA 620 TTTCAC GTGAAA 618 TGTAAG CTTACA 618 TGCAAC GTTGCA 595 GCTGAC GTCAGC 592 CCTCAT ATGAGG 583 TTTTTC GAAAAA 582 GATAAA TTTATC 559 GATTAA TTAATC 558 TCTTTC GAAAGA 537 GTTAAT ATTAAC 535 GATTAT ATAATC 533 GGTAAT ATTACC 524 TCACGA TCGTGA 519 GGTTAT ATAACC 516 CATCAC GTGATG 512 CCTCTT AAGAGG 507 GGTAAG CTTACC 478 GATAAT ATTATC 467 GATCAA TTGATC 459 GGTAAA TTTACC 454 GCTCAT ATGAGC 427 GCTTAT ATAAGC 412 TGTACC GGTACA 411 GGTTAA TTAACC 405 TTTTAC GTAAAA 402 TTTCCT AGGAAA 396 GTTTTC GAAAAC 394 GTTCCC GGGAAC 392 GCATTC GAATGC 375 CAGCTA TAGCTG 374 GCCTTC GAAGGC 371 ACCTTC GAAGGT 369 TTCACT AGTGAA 354 GTTTTT AAAAAC 341 GCTTTA TAAAGC 335 GCATAC GTATGC 328 GTTCCT AGGAAC 324 TTTCAT ATGAAA 320 GGTGAT ATCACC 320 GCCTAC GTAGGC 318 TTTAAC GTTAAA 316 CCTCTC GAGAGG 315 TATCAC GTGATA 301 CCATTC GAATGG 300 GCATTA TAATGC 297 GGCAAC GTTGCC 297 GTTAAA TTTAAC 297 AGCGTA TACGCT 296 GATGAT ATCATC 291 GCTCCC GGGAGC 285 TTTCCC GGGAAA 284 GGTACC GGTACC 281 CCCTAC GTAGGG 271 AATTAA TTAATT 269
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