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Old 07-08-2020, 01:05 PM   #1
Lechu
Junior Member
 
Location: Germany

Join Date: Aug 2017
Posts: 4
Unhappy 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


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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Old 07-09-2020, 02:38 AM   #2
GenoMax
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Location: East Coast USA

Join Date: Feb 2008
Posts: 7,076
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You have a set of indexes there that have a good number of reads. If they don't match indexes you expect then it is a different matter. There is not much you can do about the 20% reads you are losing.
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demultiplexing, illumina, indexing, miseq

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