Cells missing when I compare classical analysis VS SoloTE ?

[wjbsb]

Hi,

I test your tool on my data and I want to compare classical analysis based on genes VS output from SoloTE.

My classical analysis contains soupX treatment -> filtering -> doublets treatment -> SCTransform -> etc. After that, I have a list of "good" barcodes : so, I have 4939 cells.

I check if these barcode are present in output SoloTE after loading in a seurat object. I don't apply any filters. The dimension of this seurat object is : 18275 barcodes but the intersect between classical analysis and soloTE output found only 25 barcodes in common.

I don't understand why.

[bvaldebenitom]

Hi,

can you share a subset of the BAM file and the results obtained from SoloTE?

[wjbsb]

Yes of course...I share that next monday : I down my computer for the weekend...

What are you looking for in the bam? Any tag in particular? I launched STARsolo with maximum tags. In particular CB....

Update :

here, the first 20 rows in bam aligned by STAR 2.7.10b

A00317:401:H77CFDMXY:2:1349:13621:27555 16  1   3013768 255 88M2S   *   0   0   TAGATTAACAGATATGGAGATTTAAAAGAAATGACCAAACATCTTAAGTTAGAATCTAGGTAGTGTTATGCAAATATGCATAAATTTCGC  FFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF,FFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFF:  NH:i:1  HI:i:1  nM:i:1  AS:i:84 CR:Z:GAATCGTCAGAGAGGG   UR:Z:AGGGTTACTG GX:Z:-  GN:Z:-  sS:Z:GAATCGTCAGAGAGGGAGGGTTACTGCA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CTGCGAGCCGTACCTC   UB:Z:AGGGTTACTG
A00317:401:H77CFDMXY:2:1349:13440:27618 16  1   3013768 255 88M2S   *   0   0   TAGATTAACAGATATGGAGATTTAAAAGAAATGACCAAACATCTTAAGTTAGAATCTAGGTAGTGTTATGCAAATATGCATAAATTTCGC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  nM:i:1  AS:i:84 CR:Z:GAATCGTCAGAGAGGG   UR:Z:AGGGTTACTG GX:Z:-  GN:Z:-  sS:Z:GAATCGTCAGAGAGGGAGGGTTACTGCA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CTGCGAGCCGTACCTC   UB:Z:AGGGTTACTG
A00317:401:H77CFDMXY:2:1349:14127:28119 16  1   3013768 255 88M2S   *   0   0   TAGATTAACAGATATGGAGATTTAAAAGAAATGACCAAACATCTTAAGTTAGAATCTAGGTAGTGTTATGCAAATATGCATAAATTTCGC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  nM:i:1  AS:i:84 CR:Z:GAATCGTCAGAGAGGG   UR:Z:AGGGTTACTG GX:Z:-  GN:Z:-  sS:Z:GAATCGTCAGAGAGGGAGGGTTACTGCA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CTGCGAGCCGTACCTC   UB:Z:AGGGTTACTG
A00317:401:H77CFDMXY:2:1371:20681:34616 16  1   3014836 0   60M30S  *   0   0   CCCACCCACTCCCCCTTTTTGGCCCTGGCGTTCCCCTGTACTGGGGCTTATAAAGTTTGCCCCATGTACTCTGCGTTGATACCACTGCTT  FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFF,FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:1  nM:i:0  AS:i:59 CR:Z:CGGGTCATCGATTCCC   UR:Z:GTCAAATATA GX:Z:-  GN:Z:-  sS:Z:CGGGTCATCGATTCCCGTCAAATATATT   sQ:Z:F,,,FF:F:FFFFF:FFFFFFF,FFFFF   sM:i:0  CB:Z:CCTCTTCATTATAGTC   UB:Z:GTCAAATATA
A00317:401:H77CFDMXY:1:1436:9688:26506  272 1   3014873 1   90M *   0   0   GTACTTGGTCTTATAAAGTTTGAATGTCCTATGGGCCTCTATTTGCAGTGATGGCCGACTAGGCCATCTTCTGATACATATGCAGCTAGA  ,F:FF,:,,,,FFFFFFFFFFFFFFFF,:,FF,,,F,F:F,F:F,::FF,FF,,FFFFFFF,FFFFF,:F,:FFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:2  nM:i:6  AS:i:76 CR:Z:ACCGTTCAGAGCTTTC   UR:Z:GACAACGATG GX:Z:-  GN:Z:-  sS:Z:ACCGTTCAGAGCTTTCGACAACGATGAA   sQ:Z:FFF:FF:F,,FF,F,F:FFFFF:F::FF   sM:i:0  CB:Z:ACCCAAACAACAGCTT   UB:Z:GACAACGATG
A00317:401:H77CFDMXY:2:2377:11225:10222 272 1   3015027 0   1S89M   *   0   0   TTTAGCTCCACGGGTAATTTCTCTAGCTCCTCGATTGGGGGCCGTGTGACCCATCCAATAGCTGACTGTGATCATCCACTTATGTGTATG  ::,F,F:,F,,,:F:,::,,::FF,,F::FF,,:,:FFF:F,:FFF,FFF:F,FFFFF,FFF:FFF,,FFFFFFF,F,FF:,F:,::,F,  NH:i:5  HI:i:3  nM:i:5  AS:i:77 CR:Z:AAAGAACCAAGTTCGT   UR:Z:GGATTATTAC GX:Z:-  GN:Z:-  sS:Z:AAAGAACCAAGTTCGTGGATTATTACTC   sQ:Z::FFFFFFF:FF::FFF:,F::FFFFF:F   sM:i:0  CB:Z:AAACTGATCTCTTTCG   UB:Z:GGATTATTAC
A00317:401:H77CFDMXY:1:2124:5285:30373  16  1   3015134 3   90M *   0   0   TCACGAGATAGAGCTATATCTGGGTCCTTTAATCAAAACCTTGCTAGTGTATGCAATGGTGTCAGCATTTGGAAGCTGATTATGGGATGG  :F,F:,:F,,FF:FFFFFFFFF::F:,:F,,,,:FF:F,FFFF:FFFFFF,:F,FFFFFFFF,FF:,FFFF:F,F,F::FF,FFFF,F:,  NH:i:2  HI:i:1  nM:i:4  AS:i:80 CR:Z:TTTCCTCAGGCCCGTT   UR:Z:TTACGTATAA GX:Z:-  GN:Z:-  sS:Z:TTTCCTCAGGCCCGTTTTACGTATAAAC   sQ:Z::::FFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:TGTGCGGGTAGTGGCA   UB:Z:TTACGTATAA
A00317:401:H77CFDMXY:1:1364:11767:12070 272 1   3016274 0   90M *   0   0   CCCATATCTTCGAGGCTTTTCCCTACTTTCTCCTCTGTAAGTTTCAGTGTCTCTGGTTTTATGTGGAGTTCCTTAATCCACTTAGATTTG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:2  nM:i:0  AS:i:88 CR:Z:AGTAACCAGTAGCTCT   UR:Z:TCTCTTGAGT GX:Z:-  GN:Z:-  sS:Z:AGTAACCAGTAGCTCTTCTCTTGAGTTC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AGGCTGGGATTCGAGC   UB:Z:TCTCTTGAGT
A00317:401:H77CFDMXY:2:2160:6225:10081  272 1   3016276 0   90M *   0   0   CATATCTTCGAGGCTTTTCCCTACTTTCTCCTCTGTAAGTTTCAGTGTCTCTGGTTTTATGTGGAGTTCCTTAATCCACTTAGATTTGAC  FFFFFFFFFFFFF:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:2  nM:i:0  AS:i:88 CR:Z:CTACTATGTTCTCGTC   UR:Z:CGGGTCAGAA GX:Z:-  GN:Z:-  sS:Z:CTACTATGTTCTCGTCCGGGTCAGAATG   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CGCAGGGTGGATTTCG   UB:Z:CGGGTCAGAA
A00317:401:H77CFDMXY:1:1111:3314:20415  272 1   3017272 1   64M26S  *   0   0   CCTGATTTTAGTGGGATTGCTTCCAGCTTCTCACCATTTACTTTGATGTTGGCTATTGGTTTCCCATGTACTCTGCGTTGATACCACTGC  FFFFFFFFFFFFFFFFFF,FFFFFFFF:FFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:4  HI:i:2  nM:i:0  AS:i:62 CR:Z:GTGTGATTCGTCGGGT   UR:Z:AACAAATGTC GX:Z:-  GN:Z:-  sS:Z:GTGTGATTCGTCGGGTAACAAATGTCTT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:GGTGATTAGCGCTTCG   UB:Z:AACAAATGTC
A00317:401:H77CFDMXY:2:2320:17698:6652  272 1   3017272 1   64M26S  *   0   0   CCTGATTTTAGTGGGATTGCTTCCAGCTTCTCACCATTTACTTTGATGTTGGCTATTGGTTTCCCATGTACTCTGCGTTGATACCACTGC  FFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:4  HI:i:2  nM:i:0  AS:i:62 CR:Z:GTGTGATTCGTCGGGT   UR:Z:AACAAATGTC GX:Z:-  GN:Z:-  sS:Z:GTGTGATTCGTCGGGTAACAAATGTCTT   sQ:Z:FFFFFF:FFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:GGTGATTAGCGCTTCG   UB:Z:AACAAATGTC
A00317:401:H77CFDMXY:1:2175:29894:24956 272 1   3018512 3   90M *   0   0   TATTTATGTTGTTATTGAAGATCAGCCTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACGC  FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:2  HI:i:2  nM:i:0  AS:i:88 CR:Z:ACTGCAACACTTTAGG   UR:Z:AATGCAAGCG GX:Z:-  GN:Z:-  sS:Z:ACTGCAACACTTTAGGAATGCAAGCGTA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:ACTACGATCAACTGGG   UB:Z:AATGCAAGCG
A00317:401:H77CFDMXY:1:2175:29062:25895 272 1   3018512 3   90M *   0   0   TATTTATGTTGTTATTGAAGATCAGCCTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACGC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:2  HI:i:2  nM:i:0  AS:i:88 CR:Z:ACTGCAACACTTTAGG   UR:Z:AATGCAAGCG GX:Z:-  GN:Z:-  sS:Z:ACTGCAACACTTTAGGAATGCAAGCGTA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:ACTACGATCAACTGGG   UB:Z:AATGCAAGCG
A00317:401:H77CFDMXY:2:2249:9733:29371  272 1   3018538 1   68M22S  *   0   0   CTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACCCATGTACTCTGCGTTGATACCACTGCT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF  NH:i:3  HI:i:3  nM:i:2  AS:i:62 CR:Z:AGTAACCCAGACAAAT   UR:Z:TATACAAAGC GX:Z:-  GN:Z:-  sS:Z:AGTAACCCAGACAAATTATACAAAGCAT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AGGGAAAACACTTGGG   UB:Z:TATACAAAGC
A00317:401:H77CFDMXY:1:1143:32434:12352 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AAGTTCGCATGGAATA   UR:Z:CTCAACTAGA GX:Z:-  GN:Z:-  sS:Z:AAGTTCGCATGGAATACTCAACTAGAGC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AAGTACCGTGTGTCAG   UB:Z:CTCAACTAGA
A00317:401:H77CFDMXY:1:1143:32651:12477 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AAGTTCGCATGGAATA   UR:Z:CTCAACTAGA GX:Z:-  GN:Z:-  sS:Z:AAGTTCGCATGGAATACTCAACTAGAGC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AAGTACCGTGTGTCAG   UB:Z:CTCAACTAGA
A00317:401:H77CFDMXY:1:2162:19895:3599  16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFF:FFFFFFFFF:  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AGGAAATCATCCGGTG   UR:Z:CAAACCATAG GX:Z:-  GN:Z:-  sS:Z:AGGAAATCATCCGGTGCAAACCATAGAC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFF:FFFFF   sM:i:0  CB:Z:AGCCAATTCAGCTTCC   UB:Z:CAAACCATAG
A00317:401:H77CFDMXY:2:2223:13675:25081 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:TCGCTTGCATCTTTCA   UR:Z:TTTGTGGTTT GX:Z:-  GN:Z:-  sS:Z:TCGCTTGCATCTTTCATTTGTGGTTTGT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:TCACATATGTTGTAGG   UB:Z:TTTGTGGTTT
A00317:401:H77CFDMXY:1:1106:2636:26882  272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  FFFFFFFF:FFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:TGCGATATCGTGGTAT   UR:Z:TTGCTTATTT GX:Z:-  GN:Z:-  sS:Z:TGCGATATCGTGGTATTTGCTTATTTTT   sQ:Z::FFFFFF:FF,FFF:FFFFFF:FFFFFF   sM:i:0  CB:Z:TCCTCTTCACACCTAC   UB:Z:TTGCTTATTT
A00317:401:H77CFDMXY:1:1179:21531:21496 272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  :FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFF  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:CATTCCGCATGTTTGG   UR:Z:GCTTTATCTC GX:Z:-  GN:Z:-  sS:Z:CATTCCGCATGTTTGGGCTTTATCTCAC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CATACAGAGCTCTGTA   UB:Z:GCTTTATCTC

here, the first 20 rows in bam aligned by soloTE

A00317:401:H77CFDMXY:2:1371:20681:34616 16  1   3014836 0   60M30S  *   0   0   CCCACCCACTCCCCCTTTTTGGCCCTGGCGTTCCCCTGTACTGGGGCTTATAAAGTTTGCCCCATGTACTCTGCGTTGATACCACTGCTT  FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFF,FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:1  nM:i:0  AS:i:59 CR:Z:CGGGTCATCGATTCCC   UR:Z:GTCAAATATA sS:Z:CGGGTCATCGATTCCCGTCAAATATATT   sQ:Z:F,,,FF:F:FFFFF:FFFFFFF,FFFFF   sM:i:0  CB:Z:CCTCTTCATTATAGTC   UB:Z:GTCAAATATA GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1436:9688:26506  272 1   3014873 1   90M *   0   0   GTACTTGGTCTTATAAAGTTTGAATGTCCTATGGGCCTCTATTTGCAGTGATGGCCGACTAGGCCATCTTCTGATACATATGCAGCTAGA  ,F:FF,:,,,,FFFFFFFFFFFFFFFF,:,FF,,,F,F:F,F:F,::FF,FF,,FFFFFFF,FFFFF,:F,:FFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:2  nM:i:6  AS:i:76 CR:Z:ACCGTTCAGAGCTTTC   UR:Z:GACAACGATG sS:Z:ACCGTTCAGAGCTTTCGACAACGATGAA   sQ:Z:FFF:FF:F,,FF,F,F:FFFFF:F::FF   sM:i:0  CB:Z:ACCCAAACAACAGCTT   UB:Z:GACAACGATG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:2377:11225:10222 272 1   3015027 0   1S89M   *   0   0   TTTAGCTCCACGGGTAATTTCTCTAGCTCCTCGATTGGGGGCCGTGTGACCCATCCAATAGCTGACTGTGATCATCCACTTATGTGTATG  ::,F,F:,F,,,:F:,::,,::FF,,F::FF,,:,:FFF:F,:FFF,FFF:F,FFFFF,FFF:FFF,,FFFFFFF,F,FF:,F:,::,F,  NH:i:5  HI:i:3  nM:i:5  AS:i:77 CR:Z:AAAGAACCAAGTTCGT   UR:Z:GGATTATTAC sS:Z:AAAGAACCAAGTTCGTGGATTATTACTC   sQ:Z::FFFFFFF:FF::FFF:,F::FFFFF:F   sM:i:0  CB:Z:AAACTGATCTCTTTCG   UB:Z:GGATTATTAC GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2124:5285:30373  16  1   3015134 3   90M *   0   0   TCACGAGATAGAGCTATATCTGGGTCCTTTAATCAAAACCTTGCTAGTGTATGCAATGGTGTCAGCATTTGGAAGCTGATTATGGGATGG  :F,F:,:F,,FF:FFFFFFFFF::F:,:F,,,,:FF:F,FFFF:FFFFFF,:F,FFFFFFFF,FF:,FFFF:F,F,F::FF,FFFF,F:,  NH:i:2  HI:i:1  nM:i:4  AS:i:80 CR:Z:TTTCCTCAGGCCCGTT   UR:Z:TTACGTATAA sS:Z:TTTCCTCAGGCCCGTTTTACGTATAAAC   sQ:Z::::FFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:TGTGCGGGTAGTGGCA   UB:Z:TTACGTATAA GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1364:11767:12070 272 1   3016274 0   90M *   0   0   CCCATATCTTCGAGGCTTTTCCCTACTTTCTCCTCTGTAAGTTTCAGTGTCTCTGGTTTTATGTGGAGTTCCTTAATCCACTTAGATTTG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:2  nM:i:0  AS:i:88 CR:Z:AGTAACCAGTAGCTCT   UR:Z:TCTCTTGAGT sS:Z:AGTAACCAGTAGCTCTTCTCTTGAGTTC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AGGCTGGGATTCGAGC   UB:Z:TCTCTTGAGT GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:2160:6225:10081  272 1   3016276 0   90M *   0   0   CATATCTTCGAGGCTTTTCCCTACTTTCTCCTCTGTAAGTTTCAGTGTCTCTGGTTTTATGTGGAGTTCCTTAATCCACTTAGATTTGAC  FFFFFFFFFFFFF:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:9  HI:i:2  nM:i:0  AS:i:88 CR:Z:CTACTATGTTCTCGTC   UR:Z:CGGGTCAGAA sS:Z:CTACTATGTTCTCGTCCGGGTCAGAATG   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CGCAGGGTGGATTTCG   UB:Z:CGGGTCAGAA GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1111:3314:20415  272 1   3017272 1   64M26S  *   0   0   CCTGATTTTAGTGGGATTGCTTCCAGCTTCTCACCATTTACTTTGATGTTGGCTATTGGTTTCCCATGTACTCTGCGTTGATACCACTGC  FFFFFFFFFFFFFFFFFF,FFFFFFFF:FFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:4  HI:i:2  nM:i:0  AS:i:62 CR:Z:GTGTGATTCGTCGGGT   UR:Z:AACAAATGTC sS:Z:GTGTGATTCGTCGGGTAACAAATGTCTT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:GGTGATTAGCGCTTCG   UB:Z:AACAAATGTC GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:2320:17698:6652  272 1   3017272 1   64M26S  *   0   0   CCTGATTTTAGTGGGATTGCTTCCAGCTTCTCACCATTTACTTTGATGTTGGCTATTGGTTTCCCATGTACTCTGCGTTGATACCACTGC  FFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:4  HI:i:2  nM:i:0  AS:i:62 CR:Z:GTGTGATTCGTCGGGT   UR:Z:AACAAATGTC sS:Z:GTGTGATTCGTCGGGTAACAAATGTCTT   sQ:Z:FFFFFF:FFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:GGTGATTAGCGCTTCG   UB:Z:AACAAATGTC GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2175:29894:24956 272 1   3018512 3   90M *   0   0   TATTTATGTTGTTATTGAAGATCAGCCTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACGC  FF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:2  HI:i:2  nM:i:0  AS:i:88 CR:Z:ACTGCAACACTTTAGG   UR:Z:AATGCAAGCG sS:Z:ACTGCAACACTTTAGGAATGCAAGCGTA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:ACTACGATCAACTGGG   UB:Z:AATGCAAGCG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2175:29062:25895 272 1   3018512 3   90M *   0   0   TATTTATGTTGTTATTGAAGATCAGCCTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACGC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:2  HI:i:2  nM:i:0  AS:i:88 CR:Z:ACTGCAACACTTTAGG   UR:Z:AATGCAAGCG sS:Z:ACTGCAACACTTTAGGAATGCAAGCGTA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:ACTACGATCAACTGGG   UB:Z:AATGCAAGCG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:2249:9733:29371  272 1   3018538 1   68M22S  *   0   0   CTTAGTCCATGGTGATCTGATAGGATGCATGGGACAATTTCAATATTTTTGTATATGTTGACCCATGTACTCTGCGTTGATACCACTGCT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF  NH:i:3  HI:i:3  nM:i:2  AS:i:62 CR:Z:AGTAACCCAGACAAAT   UR:Z:TATACAAAGC sS:Z:AGTAACCCAGACAAATTATACAAAGCAT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AGGGAAAACACTTGGG   UB:Z:TATACAAAGC GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1143:32434:12352 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AAGTTCGCATGGAATA   UR:Z:CTCAACTAGA sS:Z:AAGTTCGCATGGAATACTCAACTAGAGC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AAGTACCGTGTGTCAG   UB:Z:CTCAACTAGA GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1143:32651:12477 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF:FFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AAGTTCGCATGGAATA   UR:Z:CTCAACTAGA sS:Z:AAGTTCGCATGGAATACTCAACTAGAGC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:AAGTACCGTGTGTCAG   UB:Z:CTCAACTAGA GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2162:19895:3599  16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFF:FFFFFFFFF:  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:AGGAAATCATCCGGTG   UR:Z:CAAACCATAG sS:Z:AGGAAATCATCCGGTGCAAACCATAGAC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFF:FFFFF   sM:i:0  CB:Z:AGCCAATTCAGCTTCC   UB:Z:CAAACCATAG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:2223:13675:25081 16  1   3018671 1   90M *   0   0   CTTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:TCGCTTGCATCTTTCA   UR:Z:TTTGTGGTTT sS:Z:TCGCTTGCATCTTTCATTTGTGGTTTGT   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:TCACATATGTTGTAGG   UB:Z:TTTGTGGTTT GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1106:2636:26882  272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  FFFFFFFF:FFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:TGCGATATCGTGGTAT   UR:Z:TTGCTTATTT sS:Z:TGCGATATCGTGGTATTTGCTTATTTTT   sQ:Z::FFFFFF:FF,FFF:FFFFFF:FFFFFF   sM:i:0  CB:Z:TCCTCTTCACACCTAC   UB:Z:TTGCTTATTT GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:1179:21531:21496 272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  :FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFF  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:CATTCCGCATGTTTGG   UR:Z:GCTTTATCTC sS:Z:CATTCCGCATGTTTGGGCTTTATCTCAC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CATACAGAGCTCTGTA   UB:Z:GCTTTATCTC GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2424:12798:9064  272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  FF:FFFF:FFFFFFFFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFF::FF:  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:GGGATCCAGACAACTA   UR:Z:ATGCCTTTGT sS:Z:GGGATCCAGACAACTAATGCCTTTGTAC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:GCCCAGGTACGCCTCA   UB:Z:ATGCCTTTGT GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:1:2460:32705:16767 16  1   3018672 1   90M *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCTGTTAGTTTCACTGTGTCCCTGTTTAGTTT  FFFFFFFFFF,:FFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:3  HI:i:1  nM:i:0  AS:i:88 CR:Z:CTTAGGAGTACTGCCG   UR:Z:AACTTGTGGG sS:Z:CTTAGGAGTACTGCCGAACTTGTGGGTC   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:CTCCCAATCATGGCCG   UB:Z:AACTTGTGGG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE
A00317:401:H77CFDMXY:2:1480:14597:22921 272 1   3018672 0   60M30S  *   0   0   TTTTGTTTTAGGATAAAATGTTCTGTAGATATCTGTCAAGTCCATTTGTTTCATCACTTCCCCATGTACTCTGCGTTGATACCACTGCTT  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:5  HI:i:5  nM:i:0  AS:i:59 CR:Z:ATGGAGGCATAGAAAC   UR:Z:TTCGCCATAG sS:Z:ATGGAGGCATAGAAACTTCGCCATAGCA   sQ:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFF   sM:i:0  CB:Z:ATCGTAGGTGCAGAAT   UB:Z:TTCGCCATAG GX:Z:chr1|3014749|3021108|L1MdF_I:L1:LINE|- GN:Z:SoloTE|L1MdF_I:L1:LINE

[bvaldebenitom]

Hi @wjbsb,

Please let me know when you can share the BAM file.

From the lines you posted, there doesn't seem to be an obvious error. Usually, when comparing with reference barcodes I have seen that there are differences with the "-1" that sometimes appear at the end of the tag, but that doesn't seem to be the case here.

[github-actions[bot]]

This issue is stale because it has been open for 10 days with no activity.

[github-actions[bot]]

This issue was closed because it has been inactive for 14 days since being marked as stale.