Header Leaderboard Ad

Collapse

Demultiplexing failures in MiSeq run

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

  • #2
    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.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      A Brief Overview and Common Challenges in Single-cell Sequencing Analysis
      by seqadmin


      ​​​​​​The introduction of single-cell sequencing has advanced the ability to study cell-to-cell heterogeneity. Its use has improved our understanding of somatic mutations1, cell lineages2, cellular diversity and regulation3, and development in multicellular organisms4. Single-cell sequencing encompasses hundreds of techniques with different approaches to studying the genomes, transcriptomes, epigenomes, and other omics of individual cells. The analysis of single-cell sequencing data i...

      01-24-2023, 01:19 PM
    • seqadmin
      Introduction to Single-Cell Sequencing
      by seqadmin
      Single-cell sequencing is a technique used to investigate the genome, transcriptome, epigenome, and other omics of individual cells using high-throughput sequencing. This technology has provided many scientific breakthroughs and continues to be applied across many fields, including microbiology, oncology, immunology, neurobiology, precision medicine, and stem cell research.

      The advancement of single-cell sequencing began in 2009 when Tang et al. investigated the single-cell transcriptomes
      ...
      01-09-2023, 03:10 PM

    ad_right_rmr

    Collapse
    Working...
    X