Overrepensented short sequences in the PCB114-24 data after dorado trim and pychopper

[baozg]

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Dorado version

1.0.0

Dorado subcommand

Demux, Trim, Basecaller

The issue

We are SQK-PCB114-24 to do ONT cDNA sequencing. After I use pychopper, I could alwasy see the sequence start like xxxxTTTGGGG. I cannot trim it by dorado trim and it always be soft clipped when align to reference genome. I could get 94% reads classified. Do you have suggestion to remove them?

Will dorado include pychopper step inside the demultiplexing for ONT cDNA?

Here is the command I use, to use pychopper I have to choose no-trim for basecalling. See epi2me-labs/pychopper#72

# dorado basecalling and demux
## default option will lose the primer sequences and cannot detected by pychopper
dorado basecaller -x cuda:all --estimate-poly-a --batchsize 640 --no-trim sup@v5.0.0
dorado demux -o {}_not_trim --no-trim -t 96 --kit-name SQK-PCB114-24 {}.bam


# pychopper
samtools fastq -@ ${threads} ${part}.bam|bgzip -@ ${threads} -c > ${part}.SUP.fastq.gz
pychopper -S ${part}.SUP.stats -k PCB114 -m edlib -t ${threads} ${part}.SUP.fastq.gz ${part}.SUP.chopped.fastq
bgzip -@ ${threads} ${part}.SUP.chopped.fastq

# dordo trim
dorado trim -t ${threads} -k PCB114 --emit-fastq ${part}.SUP.chopped.fastq.gz|bgzip -@ ${threads} -c > ${part}.SUP.chopped.trimmed.fastq.gz

The example reads after all process

109:800|6d3aa2fe-233c-4bc6-954d-2ef65bdb19fd    GCGCTTCGGGTTCGAGTTGCACTTTGGGGATCTTCATCAAATTCTCTCTTTCATCTACAATCACTCATCAATGGCGATTTCTCAAGCTCAGCCTCTGCTTCTTCTCCTCTTATCACTCTTCTTCTTACCTGCTTTACACGCAACTTCTTTCTCCTACTGCGATAAGAGGCTCGATCCCGTTAAGGTCACGGGTGTGAAGATCTCTC>
103:462|4dca35f1-7592-431b-b77e-64a8cd61769a    GGAATTAGACTTCACCTTGGCGTTTGGGGGCTGATTCTTGTAGAGAGCAGAATCCATCGTCTTGCTCGTTACAAGAAGACCAAGAAGCTCCCACCCGTCTGGAAGTACGAGTCTACAACTGCAAGCACTCTTGTTGCTTGAGAAGAATAGAGTTGATCATGTCCTTCAAGAAGGACCATTTCATTGTCTGCATTGCAACTCAAAGC>
111:892|3bb42dd6-6a9f-41d8-8422-ab6d7baaa9c5    CCGGTTCCCCTTCGCCTTCGGGTTTGGGGAAGTGAGTAAGAGAAAAGAGCAAAAGAAGAAGAGAAACAACAAGAAGTAGTAATGGCTTCCTCCATGCTCTCCTCGGCCGCTGTGGTTACCTCCCCGGCTCAAGCCACCATGGTCGCTCCATTCACCGGCTTGAAGTCATCCGCTGCTTTCCCGGTCACCCGCAAGGCCAACAACGA>
129:584|ba6678c8-f2e8-410d-97db-ab71d844bdab    AGAATTAACGTTCGAGTTGGCCTTTGGGATCAAACGTTTGTGTTTTGCATTTGCTCTTACGTCACTTGATTTACAATTTCCTTTCCCCATTATTTTCTTATAAAGAACTATGATTTCAGTTACTGAATTTATCCTCTGCATAGATGACAATGTTGGTGGTACGTGCATTGGAGGCGAGGTCGTTATCAGTGGTCAAGCATTTGTGT>
132:596|6cc8965f-cd8c-488f-a15e-2cacc1b56fc6    CGAATTAAGGTCGAATTCCAGTTTGGGGAGGAAGTATCAATCGAATCAGACATCGACGAGGAGCTTCGGTCTTTAAATCCGAGGAAGAGACTAAAATCGGATAGTTGCGAAAAATGCCGATCATGGAGAAATTGAGGATGTTCGTGGCGCAGGAACCAGTTGTTGCTGCTTCTTGCTTAATCGGCGGTGTTGGACTATTTTTGCCT>
106:606|7fc8ebd8-bde1-490e-878e-7b6760c2a77a    CCCACTTCCCCTTGCGCTTAGGCTTTGGGAAACGTTTGTGTTTTGCATTTGCTCTTACGTCACTTGATTTACAATTTCCTTTCCCCATTATTTTCTTATAAAGAACTATGATTTCAGTTACTGAATTTATTCTCTGCATAGATGACAATGTTGGTGGTACGTGCATTGGAGGCGAGGTCGTTATCAGTGGTCAAGCATTTGTGTAT>
128:1049|014488b1-f2e5-43d3-9810-85d1c2fe57d7   AAGGTTACCATTACGATTCCGATTTGGGGGTGCCCTCACATCTCTTCGTCTTCTCCGCCTCACTCCGATCTCTCTACGATTCATTCTTCAATGGCTTCCATTGCTGCTTCTGCTTCTACTTCCCTACAAGCTCGTCCTCGCCAACTGGCGATTGCCGCTAATCAAGTTAAATGCTTTAGCTATGGAAGAAGAAGCCATCGTTCTTT>
126:796|4175fb70-24dc-4d46-bb15-d79ba56852e0    GGGCTTCCAGTTAGCGTTAAAGTTTGGGGAAGTAAATTAAGATGGCATCTTCTGTAGTGTCTTCTGTGTTTCTCTTTCTCCTTCTTCTTCTTGTATTTCCACATATAGATAATGTCCTTGGCTCACGAATGGAACTCCGTGAACTTGAGGAGATAAACCATGCTGATCCAGTGACGGTCCAAAAGCGCTCGATTCCTTTTTTACCG>
110:849|328989cd-ac66-45e1-a8a8-2dd7a2959bd8    AACGTTAGCATTCGCATTAGAGTTTGGGGGTTGCCTCAAAAGATGCAGATCTTCGTGAAAACCCTAACGGGGAAGACCATAACCCTAGAGGTTGAATCATCCGACACCATCGACAATGTGAAAGCGAAGATCCAAGACAAGGAAGGAATCCCACCGGACCAGCAGCGATTGATTTTCGCCGGGAAACAGCTGGAAGACGGCCGTAC>
97:909|4df1fc7c-ddec-4989-94bd-42ab6f37aa9b     CAGGTTGGGGTTAAAGTTGCGATTTGGGGCTCCTCTTTCTCTCTGAAACTAGATTGGGAACCATCACAACCAAAACCAATTTGTTTGTCTCTTCTTTCACTCTCGATCTCTCTCGATCCCTCTGAGTCTTAATTCCTCTCAGTCTCAACTCTGGATCTCGGTCGTCAGTTTCATCGATCTACAAATCTCTCTTAATCGTTTCTACT>
109:461|a68923af-1bdc-4e9b-a46b-f4adf0b95a2f    CAAGTTCAGGTTAACCTTAAAGTTTGGGGGACAATGTGGAAGCTTCCATTGTTCGGATGCACCGACTCCGCTCAAGTGTTGAAGGAAGTGCAAGAATGCAAGAAGGAGTACCCTAATGCCTTCATTAGGATCATCGGATTCGACAACAACCGTCAAGTCCAATGCATCAGTTTCATCGCCTACAAGCCCCCAAGCTTCACCGATGC>
126:1098|f97bc33e-1dcc-401b-8e98-7f64a64ad8ce   GGCCTTGGAATTACCGTTAGAGTTTGGGGGTTTTCTTTAGTGTGTGATCAAAAAAGATCCTTACTTTTTTTACAGAATCAGATCTTTGAAAGAGTTTTGCTCCTTGATACATATCGTCAATGGCTTCTCCTATTGTTGTAGAAGATCGTCCAGCTCCGCTCGATGCTACAGCTGATCCACCGGCTCTGTTCGACGGAAGCACTAGA>
104:1141|725193ce-f86b-41ab-8c15-73af71870a52   GGACTTCACCTTACACTTCGACTTTGGGATCTACTTCTTCCACATCTCTGCTTCGATTTGGTTTAGGTATTTTTAGTCTTTTGGTTTTGTAAAAGTAAGTTTCAAACGAAAAATATTACCTGGTAAAAAAAAAGGCCAATCGTTCCCGTAGCATATCCTCTCTTCTCTCTCCTTGAAGACGATTTGTCTGGTTTCGTTTTTTTAGA>
137:728|e1a44f29-2566-430c-af08-051bac4ce665    AGGATTGACCTTAGCATTGGACTTTGGGGGATTGGTAAAGCAGAATCCCTAATTCATCAGCGAGACGCAGGAGCAAGATGATGAAGCACACGCAAAAGATTGTCGCAAAGTACTGTGGAAGAGCGTATTCAACCATGGCATCTTCCCCACTGTTCTCGAGCGTCAAGCATGGGTGGAAGGAGCAGGACGGCTTGGTTCTTGCTGTG>
101:899|cd4467df-3f57-4154-b4bb-f3bbd6a2c6c8    ACGGTTACAGTTCACATTCGGCTTTGGGGGTGTTCTTCTGGTTCTCCCCAATATTGTCAGTATGCTGACAACAAAATTATTGTAACGAGGTTCTGAATTGGCTAAGTGAAGCAGAGGTAGACCGGCTCCTTTCTCCGGTAACGGCGAGGTGTGGTATCCATCGGCGACGCAGCTGTGGCTCTTTCACGGTGGTCGAGAAGCTTCTC>
106:870|ae8453ab-576a-445d-a023-e3ee6ee7cf8e    CGGATTCAAGTTAACATTCGCATTTGGGGAAGTGAGTAAGAGAAAAGAGCAAAAGAAGAAGAGAAACAACAAGAAGTAGTAATGGCTTCCTCTATGCTCTCCTCGGCCGCTGTGGTTACCTCCCCGGCTCAAGCCACCATGGTCGCTCCATTCACCGGCTTGAAGTCATCCGCTGCTTTCCCGGTCACCCGCAAGGCCAACAACGA>
134:738|b18a13a5-634b-4a68-9295-2be1fb271953    AGGATTGCACTTCGGATTAGAATTTGGGGAGCTTAAGCAAATTCAAAGCAACACTAGAAACCCTAGCCTCTCTCTTTTATATCTCACAACAAATCTCTTTACTATTTGAAATGGCGATCTCTAAAGCTTCCATTGTTGTTCTCATGATGGTGATCATCTCCGTCGTTGCTTCAGCTCAATCTGAGGCACCAGCACCAAGTCCTACA>
112:1450|20ac9f34-e804-4417-8c8e-48030d96f758   CAACTTCAAGTTCAAGTTCCAATTTGGGGGACTCAACTCAAGAGTGAAGAAAGAAAGAGACAAAAAAAAAATGGGAGGCAAAGGTGTGATGATGGTGGCGATACTTTGCCTTTGGGCACTCTCTGCAACATCTGAAGCAGTAACAGAGGAGGCTCCAGGTTTGATGATGAACTTCTACAAGGACACGTGTCCTCAGGCCGAAGACA>
111:910|37587b02-94a2-412a-bba2-5ae2eb61f8ef    CAAGTTGGCGTTAGGGTTAACCTTTGGGATTTTCGATTTGATCTTCCTTCAATGGCTGTCGTCGGCGCTCCGATATCGTCTCCAGCGACTCACCTGCAAACAAAATTTATCTCCAATCCCATTCTCCCTCGCTTGCGCCGGTCTTTCTCCACCGGAAAAGCACCATCAACTTTCTCCGTCGTCTCTATGGCTCCCCAGAAAAAGGT>
127:1278|b45932ff-f13c-47d2-ab40-9f8f77e9ce7b   AAAGTTAGCCTTAAGCTTCACGTTTGGGGGTCTTCTCGTCACTCTCAATCTCACAGGCAAAGCCTCTCGTTGCTAGAGGAATCAAACAATGGTGTTTGTGAAGTCCTCCAAGTCGAATGCTTACTTCAAGAGGTACCAAGTTAAGTTCAGGAGAAGAAGAGATGGGAAGACTGATTACAGGGCAAGGATCCGTCTTATCAATCAGG>
106:637|fd078da4-1ffd-4975-b89e-3250dda0c78e    ACGGTTGCGATTGGGGTTAGGGCTTGGGGATTCCATACTCTCTTGAGTCTTGTCGTAGCAGAATCTCTGATAAGCTAACCAAAATGGTCCTTCAAAACGATATTGATCTGCTTAACCCACCTGCTGAGCTTGAGAAGAGGAAGCACAAGCTTAAGCGTCTTGTTCAGTCTCCCAATTCATTTTTCATGGATGTCAAGTGTCAAGGC>
128:860|8627e41b-c842-41b4-8a4e-f797f72ecf23    CTGAGCTTCGCCTTGAAATTAGACTTTGGGGAAGTTCTGTTCTGGTTGTCGTGTGTTTTGTCTAAGGGAGAAGCGAGGGAAGAGATTAAAAGATTAAACCTTTTTCGCCTTCATGGATTCGAAGTTGAGGAATTTCGTGATGGTTCTGTTTGTGATGACTGCGATCTTGAAAGGTTCTGGTGTCGACGTGTGGACTGGCGAGATTC>
110:751|ec94dcad-f9ed-49b7-a306-94293bd42d59    ACAGTTGAACTTCAGGTTGACATTTGGGGAGACTTAAGTTTTTTCATAGTTTGAGAAATTTCGCCGCCGTTTGAAGAAGAGATGGGAGTTTTCACTTTCGTTTGCAAAAGCAAAGGCGGAGAATGGACCGCAAAGCAACACGAAGGACATCTTGAAGCTTCAGCTTCTTCTACTTACGATCTCCAGCGCAAGCTTGTTCAGACTGC>
141:233|ef9fd1c9-5f2d-4b05-abbf-99981313e604    GAGATTCCGCTTGGGCTTAAACTTTGGGAGACATTTTCTATCGGATTCTCAACTGTCTGATGAAATAAATATGTAATGTTCTATAAAAAAAA    RLNJMSJKKNMLKQSSQKNLJJIMHSOMSRSPSPSSSSSSPPSQSSSSSRQRNLJLNJJLDESSMIOPKGIIHHMKA>=4/.-A=57;;30,
112:822|c23a9cf1-fe70-487a-a94e-bc06e942aeb4    GCAGTTAAGGTTCAGGTTACAATTTGGGGATCCATTCTCTTTTTTTGCATGTTTGTGTTGTAAAGCAGTGGACAGAGAAGACGATGAGTACGATGAAGCCAAGTCGGAGCGACGAAATATCCGACCCGGATCAACAGATCAAGAACGCGAACCAGATCAGAGCTGATTTCGATTCCTTGGCTCCCAAACGACCCACTAAACCCACT>
131:938|7bcde823-c655-4ff0-8d52-f23472727331    AAACTTACAATTCAACTTAACATTTGGGGGCTCGGAGGATCAAAAGACTCAAATCAGCAATGTGGAGCGTCACCGGAGCATTGACTGTGGCGGTGCCGCCTACCGCCGCCGCTTGCCGTCCGAAACCCTTTTTCGTCTCCTCTTCCTTTCCCAAGCAGACAAAGAAACTCCATCTCTCTCCGCCGTCTCTGTCTCTACCATCATCA>
133:1202|339b5791-e7c6-4965-9f34-3b10430729dd   CGCCTTAGCGTTGCAGTTCAGGTTTGGGAGCTCTTCCTAGAATTAAACCAGAGTAAAGCTTGAAAGTTGACACTTTGGGCTAAAAGAATGGCGTCTTTGGGTGTGTCGGAAATGCTTGGTACTCCTCTTAACTTCAGGGCAGTTTCAAGATCGTCTGCTCCGTTGGCATCAAGCCCGGCCACGTTCAAGACAGTTGCTCTGTTCTC>
113:521|fb28013d-cbee-4dd6-827f-a0bb797a4122    GCGATTGAGCTTAACCTTACGCTTTGGGGAGGGGAAAGGTTCCATGTGAACGGCACTTGCACATGGGTTAGTCGATCCTAAGAGTCGGGGGAAACCCGTCTGATAGCGCTTAAGCGCGAACTTCGAAAGGGGATCCGGTTAAAATTCCGGAACCGGGACGTGGCGGTTGACGGCAACGTTAGGGAGTCCGGAGACGTCGGCGGGGG>
101:860|bb9a6675-d562-4e35-a8fd-b07483b609b5    CCCGTTCGAGTTCGCGTTCACCTTTGGGAGTCACACAAAGAGTAAAGAAGAACAATGGCTTCCTCTATGCTCTCTTCCGCTACTATGGTTGCCTCTCCGGCTCAGGCCACAATGGTCGCTCCTTTCACCGGACTTAAGTCCTCCGCTGCCTTCCCAGCCACCCGCAAGGCCAACAACGACATTACTTCCATCACAAGCAACGGCGG>
131:1067|347d9a06-b6bc-41bf-8ca0-e7d454e3ac20   GCCATTGGCGTTCAGATTCCGGTTTGGGGAACAAAGCTAACCAAACAACTGTGCGAGTAAAGAGAGAAGAAAGAGAGAAAAAGCTAAAGATGGTGCTAAAGGTGTACGGACCCCACTTTGCTTCACCAAAGCGAGCTTTGGTAACGTTGATCGAGAAGGGCGTTGCCTTCGAGACCATCCCCGTCGATCTCATGAAAGGAGAACAC>
130:873|56586cd8-47d1-4e5a-989b-f8091d9b9b18    ACGGTTGCCCTTAACATTCAGATTTGGGATTGAAGAAAAAGCTTTGACAAAACCAGAGAAAGAGACAGAGAGATAATGGCGGCTAAACTAATCTGTTCGTCTTTAACAGTGCATTCAATGGCGAACAAGAAACCTTCACCTTCTGCTGCAACAAGAACAATAACCTCAAAGAAGAGTACAGCGACTCCACAGGTGAAACTACTGAC>
103:865|41fd20c5-08a1-4c8f-a46e-c5e4630894f7    GGGTTACCATTAAGCTTCGGGTTTGGGGGAGATCTCTGGCTTCCCCTCCCCCTCTCTTAGCCATGGCTGGCTTTCATTTGTACCACTCGTCCTCCGGTTTCTCTCTTAGCCCTGCCATATAACTCTTCTTGTTCGATTTTGTTTGATTTACTTGGTTCTCGGTTTCTTATGGGATTTTTTTGTTCTCAATCTCCTGGAGAAGTATG>
125:927|afc4cf78-7d8f-4e74-b23d-60b68a394f4f    CGGGTTGCGATTAGCCTTGCGGTTTGGGAGCACACAACTCTAACCGTTTTTTTTCTCAATCTCTCAAATCTCAAAACTCTTCAACAATGTCATCGAAGAACGGAGTTGTTCGAGCCGTTTAGGATCAATGGACCACATCAAAAAAGTCTTCCAAAGATTCGACAAAAACGGCGACGGAAAAATCTCCGTCGACGAGCTAAAAGAAG>
128:1134|79e878c9-508b-4e1d-a590-e6d7a06aded7   CAAGTTCAGGTTAAAGTTCACCTTTGGAGCGGGAACAACGCTTTGACGACGCATCGGTTCTGTTCGCCTAGCTTCTTGATTTCTTCTCCTTGTTTCATCGGCTTGACCGGAATGGGCTCTGCTACTCAGTTACGTGCTCGTCGTTCTCTGATCTCTTCCGCAGTTGCGACGAATTCGATGTTGCATGACGTCGGAGCCACCGTGGC>
108:367|f8f9239e-5856-4819-b4fd-d1d52c2ab697    GCAATTGAAGCTCGACTTGGGATTTGGGAGGAACCAAGAAAGACAACACTAACTTGCCATCCGAGCGCTCAATGACACAGAATGGTTATGCTGCCATTGAATAGAACTTGTGTTCCAGCGTTTACGCCTTTTATATGTAGCTACAACCATATATCGTTGCATTTCAAGTGTTTGTACCATTTCTGTGATAGACTTTTTGGACTAAT>
105:1155|175f3101-d435-4035-bb58-05d65d4d3282   CGGGTTCAACTTACCATTACAGTTTGGGGACTCTCACCAGTCACAACAAAAAACAAAACAGAAAAACAAAAAAAAGATTCTAATGGCTTCCTCCACCATGGCTTTGTCCTCCCCTGCCTTCGCCGGAAAGGCTGTGAAGCCGGCGGCATCAGATGTCCTCGGAAGCGGCCGTGTGACCATGAGGAAGACTGTCGCCAAGCCAAAGG>
125:561|f36d1a48-ad98-4f29-ae77-2cef5edb3fb2    AACCTTCCGATTCCCGTTAAACTTTGGGAGGAGTCTCGGAGCTGCCATCATCTACAACAAGGGTCATGCTTGGGATGACCACTGGATCTTCTGGGTCGGTCCATTCATCGGTGCTGCGCTTGCTGCTCTCTACCATCAGATAGTCATCAGAGCTATTCCTTTCAAGTCCAAGACATAAAGCTTCCTACATATTCTCATCATCATCA>
106:937|c8438257-721e-4f52-9092-c92be08d6b5a    AAGCTTAACCTTGGAATTGCCTTTGGGGACACAAAGAGTAAAGAAGAACAATGGCTTCCTCTATGCTCTCTTCCGCTACTATGGTTGCCTCTCCGGCTCAGGCCACAATGGTCGCTCCTTTCACCGGACTTAAGTCCTCCGCTGCCTTCCCAGCCACCCGCAGGGCCAACAACGACATTACTTCCATCACAAGCAACGGCGGAAGA>
119:1132|8ffda615-86e1-401e-b9d0-24ef452bc14e   GCGCTTGCCGTTCAACTTAAGATTTGGGGCTTGCCTTCTTGTGTCACTCATCTTCACAGCAAACAATGGTGTTTGTGAAGTCCTCCAAATCGAATGCTTACTTCAAGAGGTACCAAGTAAAGTTCAGGAGAAGGAGAGATGGAAAGACTGATTACAGGGCCAGGATTCGTCTTATCAATCAAGATAAGAACAAGTACAACACGCCT>
128:1261|af266241-7895-4b62-ab35-c8be051dee43   ACGCTTCCCCTTGCAGTTGGGATTTGGGGATAACCAGACACGCACCCATTTTTTTGTCTGAAGCTTTTAGCGAATTTGCGTGTGTGCGTGGTTATGGAGTCTCCGTTCAAACCTGATGTCGTCAGAGGTCAAGTGGCTCTCATCACCGGTGGTGGATCCGGCATCGGTTTTGAGATCTCTTCTCAGTTTGGCAAACATGGAGCTTC>
120:1175|9439b1d3-e060-42bc-bfba-e8495f4cd5a6   GGCCTTAAAATTCACATTACACTTTGGGGAATCTCTTTACTTGCCACACCACAACACATCACTGATCCTTCTCTCATCCTCAGACGCAGCAGCACAAACACAAACCCAAAATATCTCTTTAATGGCTCAAACCATGCTGCTTACTTCGGGCGTCTCCGCCGGCCAATTCTTGAGGAACAAGAGCCCTTTGGCTCAGCCCAAAGTTC>
133:760|5d4b4224-6085-44e2-b4d3-979fa9b6589b    AGACTTCGGGTGGCATTCACGTTTGGGGATCTAAGCGAAGGAAAAATGCCAGCGGGTCATGGAGTTCGGGCGAGAACGAGGGATCTGTTCGCGAGGCCATTCAGGAAGAAGGGTTACATTCCACTATCGACTTACCTGAGGACCTTCAAGGTCGGCGATTACGTCGATGTGAAGGTAAATGGTGCGATCCACAAGGGTATGCCTCA>
128:1156|3f2051ae-aaf1-4e3e-8ca4-1e7b24de06a1   CGAATTAAAGTTCAACTTGCGATTTGGGGATCCTCAGACGCAGCAGCACAAACACAAACCCAAAATATCTCTTTAATGGCTCAAACCATGCTGCTTACTTCGGGCGTCTCCGCCGGCCAATTCTTGAGGAACAAGAGCCCTTTGGCTCAGCCCAAAGTTCACCATCTCTTCCTCTCTGAAACTCTCCGGTTGTTCTACCATCTCGG>
140:764|51205691-cfea-4ba7-8488-90e5bff86313    GCCCTTCCCGTTCCCATTACAGTTTGGGAGAAGAAGAGTTTGATCATCTTGTGAGAAAATAATGGCTGCTTCAGTGATGCTTTCATCGGTGACATTGAAACCAGCTGGTTTCACGGTGGAGAAGGCGGCGGCTAGAGGATTACCGTCGCTCACAAGAGCTCGTCCCTCATTCAAAATCGTCGCCAGTGGCGTCAAGAAGATCAAGA>
98:1119|7b17fed1-3a19-4b17-a883-6015b96c86e2    GCGCTTAAACTTCAACTTCCAATTTGGGGTTGTTGTTTAGTCTTCTAGATCTGTAGTTTTTTTTTCCGCGACCGGCCATAAAGTTTATCTAATCGGGCGGTCGATTGGATCGGAGATTTAGGTAAAGAGAAAATGGCGCAGCAATCGTTGATCTACAGTTTCGTCGCTCGCGGCACGGTGATCCTCGTTGAGTTTACTGATTTCAA>
114:796|07636863-3147-45a6-9654-bae503b9ec52    AACCTTGGCCTTACCACTTGCACTTTGGGGACTTCAAATCCATAATCTCGCTACTTCTCTCTCTAGATCCGAAAAATGGCGAAACTGGTGATGTTGTTTTTGGTTCTCTGTATCTTGCCGGCGATAGCCATGGCGGCAAGGAGGGGAAATATTGGAAAGAATACAATGGTGGTTCAAGGTCGTACTTACTGTGACCTTTGCAAATT>
131:1425|3b242f28-2a43-4c1e-8e87-f34eb67c5bae   ACAGTTGACGTTACGCTTAACCTTTGGGGACTTCCACAGTTTTGAGTTTTGAAACTTTGACGGCTCGACAAAAAAAAAGACGCCATTTTTATTTCCTTATTTTAACAGAGAGAAATCAAAGAGACAAAGCCACGATCCGCCATAGCTGTTGTTTGAGATCGATCCCCACCAAAGGAGCCTAAATCATGCCTTCAGGTGCTAAGAAA>
129:1306|1613e1ec-bf3f-49dc-83ea-af6ba8bb8c16   ACCGTTACGGTTAGCGTTACCGTTTGGGGGTTTAGCTTTTTCGTTCCGTACATCTTCTTCTTCTTCTTCTTCCCAGAAATATGGCTGAGGCTTCTGACTTGTTGGAATGGCCTAAGAAGGACAACCGTCGTTTTCTCCATGTTGTTTACCGCGTTGGTGATCTTGATCGTACCATTGAGTTTTACACTGAGTGTTTTGGTATGAAG>
135:933|c94da89d-3a7a-4385-baee-f42a46cf9e59    AACCTTACCGTTCACGTTGAGCTTTGGGAGTCACACAAAGAGTAAAGAAGAACAATGGCTTCCTCTATGCTCTTCTGCTACTATGGTTGCCTCTCCGGCTCAGGCCACAATGGTCGCTCCTTTCACCGGACTTAAGTCCTCCGCTGCCTTCCCAGCCACCCGCAAGGCCAACAACGACATTACTTCCATCACAAGCAACGGCGGAA>
106:750|0ed56114-9a38-4ebd-9825-f3ca5d875343    ACACTTCCACTTCGGCTTGACATTTGGGGATCGAGTGATAGAGGAAAGAAGAAGAAGAAGAAAATGCCGTGTCTCAACCTCTCCACCAACGTTAACCTTGACGGCGTCGATACATCTTCCATTCTCTCGGAAGCTTCCGCCACCGTCGCCAAAATCATCGGCAAGCCTGAGAACTATGTGATGATTGTCTTGAAAGGCTCTGTGCC>
107:1189|799a58ee-a7eb-48a8-9092-7e2f82773ba4   GCCCTTAACCTTCGGCTTGCAATTTGGGGATCACAAATTAAACAATAGACCATACCACAAAAAAAAAAACATGAAAATCCTCTCACTTTCACTTCTCTTTCTCTTTGGCAGCTACGGTCTCCCACGTCCGGTCTTCGGCTTCCGTTCCAGGGCTCATCGAACTCCTCGAATCGAACACCATCTTTGGGGACGAAGCCGAACTCTAG>
125:764|fb6c9dde-6901-413c-bacc-2e6b247d7219    CCAGTTCCGGTTGCAATTCCGATTTGGGGTTCGTTCTGTTTGTTTTTTTATTCGAAGAGATGTCTGTCTTCGAAGACGATTCCTTCGTCATACTCAACGATGATGCGTCTGAGTCTGTTCCAGTCTCAGGCTCTTTTGACGCCATTGACTCTTTCTCAGCCTACGACGGCTCAGTCCAAGTCGATGACTCCGTCGACGATGTTTTT>
117:1484|2a1525a8-091a-49d5-9250-da6efe446e92   CACATTCAAGTTAGAGTTAGCCTTTGGGATTTTCACATTCTTCTCTCTCGATATCATCTAAATCTCTCTCGATCTCAATTTCGAAAAATGGCTGACAAGAAGATCAGAATCGGAATCAACGGTTTCGGAAGAATCGGTCGTTTGGTTGCTAGAGTTGTTCTTCAGAGGGATGATGTTGAGCTCGTCGCTGTCAACGATCCTTTCAT>
116:852|155e53a1-4fcc-4d8a-b4e6-ed42eeda54dd    CAGATTGAGATTGAGGTTCAGCTTTGGGAGACACAAAGCACCGAAAATGGCGGTTCTTTCTGGAACATTCTCTACCTTCCTCTGCATCAAAACGACATCTTTCCACAACCCATCAACTCCTCCTTCGTCTCTCCGGCTAGCCTCTAACCCGGCGGCTAAACTACGCTTGACTCGCGCGGTTACATCTGCGACGGCCTCTTCAGAAC>
148:643|bacc02e6-45e5-4857-ac3c-f563e11a3b5d    CGAGTTCACATTGCGGTTAACATTTGGGGTAGCGGGTATAGTCTAGTGGTAAAAGTGTGATTCGTTCCTTTAATCCCTTTAATAGTTAAAGGGTCTCTCGGTTTTATTCATCTTCCGATCAAAAATTTTATTTCTTAAAAGGATTTAGTCCTTTCCCTTTCAATGAAAGATTCAAGGAAGATTATAGATTCTCGTAATTTGTATCC>
105:649|425e76de-3912-464c-a44f-7b62bef0386f    AGCATTAAGGTTGAACTTCGGGTTTGGGGGTCGAAACGCCGTAGACGCCAACACGCCGGAATCGTATCCTATCCTTTCTTCTGATCCTTACCAGAGAGCTCATGCTACATTCTGGGCTGATTTCTTCGACAAAAAGATAAAACAACTGATGATGTTGAGGTCATAGAAGATTGGGAAGAAGAAGAAGAGAATGAATCAAATGTCAC>
98:650|cb72ff9d-6dcc-4efb-9edc-b88a20b4c79c     CAAATTGACATTACGGTTAAGGTTTGGGATCAGAGATTCGTCGATCGAAAATGAATACTGACATCACTGCCTTGGAGAAGCCACAATACCCAGTCGTTGATCGGAACCCTGCTTTTACTAAAGTCGTCGGCAATTTCAGCACTCTCGATTACCTCCGTTTCTCTACCATCACCGGCATCTCCGTCACCGTCGGCTATCTCTCTGGG>
136:1001|e1c81bb4-bb63-45d6-8c88-9ab94f0993df   GACGTTAAGATTCCACTTCGGGTTTGGGGAAGGAGAAGCCGCAAACCATTTTTTGTCAATGAGGGTTCTTCTTCGTTGGAGATTTCGCTTCCGTTTTCACTAATCGATCTGTGAAGATGTTCTCCGCTCAGAACAAGATCAAGAAGGACAAGAATGCTGAGCCAACAGAATGCGAGGAGCAAGTTGCTCAGGCTTTGTTTGATTTG>
152:1031|fe23db2a-dba8-458a-8955-b7cb8e70ee33   AGCATTACGATTGGGCTTGGGCTTTGGGGATTTGCGATCAAGCAATACTCTCTCTCTTTCTTCTGCCATCGTCAAAAACTTGTCTTTTTGCTTCTGGGTTTTGTTTCTTATTTGGAAATAGTCATCTATTCCGGGAAATTTGGAGATGGCTCCGGTTGGTCCTCGATCCGGTGATGCGATCTTCTCTAGCATTGATCATGTGAATG>
130:1248|ffb00bda-28bb-47c1-bc65-1229febb1c71   CGCCTTCGACTTCAACTTGGGGTTCTGTATCGCCATTTTTTTTTGTTGGAGAGTGAAGAAGGAGTAGGATTCAGAGGATGTATGCTGATAAACCCTACTCTTCTTTCCTTCTTACCTCCAACTCTTGCATTTCATCAGCCCTTGTTCTGTAAGGCATTTTAGTTGGTAAGGACTAGAATTGGGTGCAGCACGCCAAGTGCACATGT

### System specifications

Linux, GPU A100, run in CentOS 

[malton-ont]

Dorado currently performs strand orientation and UMI detection when trimming primers. This occurs in either the basecall command or in the trim command. Note that dorado demux does not trim primers explicitly (only barcodes, and then only when classifying), and so does not include pychopper functionality.

You should be able to do everything you are attempting in a single dorado call:

dorado basecaller -x cuda:all --estimate-poly-a --batchsize 640 sup@v5.0.0 --kit-name SQK-PCB114-24

I'm not clear where your sequence fragments are coming from. They don't appear to be part of any ONT barcode or primer that I can see - are you using custom primers? I also don't see PCB114 in the pychopper primer data or the available kit arguments - I assume you've added this in yourself?

[baozg]

Thank you! I will run the dorado basecaller only and see if the strand was correct or not. The last time I have experience with dordao basecaller from the seuqenicng providerer don't give me the correct orieation.

pychopper do have option for PCB114, but it basically use PCS114 primers data,see code from this repo from epi2me. Old pychopper already in archive.

[malton-ont]

Ah, thank you - I'm not on the epi2me/pychopper team so I was looking at the wrong repo!

[baozg]

I have tried the dorado basecaller only, it didn't orientation the cDNA reads by primer direction. So it don't have consistent output with pychopper. You could see the strand by color option in IGV in PacBio IsoSeq and reference annotation.

If I did all the step, the strand is correct, just have these xxxTTTGGG sequences in the cDNA reads. The default trimming not compatiable with pychopper in the epi2me epi2me-labs/pychopper#72

[malton-ont]

@baozg,

Strand orientation was introduced in dorado-0.9.5. I believe the strand orientation is only stored in BAM files, and you look to be generating fastq?

I'm not familiar with using pychopper, but my understanding is that it also trims reads? If pychopper has already performed trimming, dorado is not going to find primers to trim.

Does dorado trim the reads correctly if you haven't passed them to pychopper? As I said, those fragments don't look to be part of any barcode/primer kit that I can see in dorado. What do the reads look like before you trim them? Are you able to share any data?

[baozg]

If I understand correctly, I should use the tag in the BAM to perform reverse complement by myself? I will look at this result with more detail. Pychopper do also trim the primers in the sequences.

My workflow follow dorado basecaller --no-trim + dorado demux --no-trim + pychopper (primers) + dorado trim. I could upload part of reads with dorado basecaller / dorado basecaller --no-trim + dorado demux --no-trim + pychopper (primers)

[malton-ont]

@baozg,

I'll say again - I don't recognise that fragment as part of any of our kits. Are you able to identify where it is coming from?

Are you using custom primers? If you are, you should be using the --primer-sequences parameter to set these up for trimming (or modifying pychopper to do this for you).

If pychopper is trimming the primers itself, the primers are not there for dorado trim to remove. Whatever is being left over is therefore a result of pychopper.

[kdolan1973]

The SQK-PCB114-24 sequencing kit is associated in dorado with the PCS primers, so as long as the sequencing-kit field in the input pod5 file is set correctly, running dorado basecaller with demuxing and trimming enabled should correctly detect and trim the PCS primers, and infer the strand orientation.

When running dorado trim, the -k option needs to be the sequencing kit used (again SQK-PCB114-24). It won't recognize just PCB114.

Note that for the dorado basecaller command, if --no-trim is specified it will not attempt to detect any primers, or to classify the strand orientation. So, your two options are:

1. Run the entire process with a single dorado basecaller command, with trimming enabled. If the sequencing kit is specified correctly in the pod5 file, this should just work.
2. Run dorado basecaller with --no-trim, then run dorado trim with -k SQK-PCB114-24. This should work even if the sequencing kit is not correctly set in the pod5 file.

If neither of these works, then we will need more information to debug the issue. Also note that while dorado will report the strand orientation in the bam file (if it can), it will not reverse-complement the called sequence for reverse strands. You will need to do that separately. This is because many other downstream tools would not function properly if dorado automatically reversed those reads.

[kdolan1973]

It also looks like the bits of sequence in your examples above that come before the TTTGGG sequence are the UMI tags. I think what is happening is that when you ran pychopper in your workflow, it trimmed the primers, but not the UMI tags.

If you run dorado basecaller to do the complete process, is the only issue that it doesn't reverse the strands that have been classified as reverse orientation? If so, then the proper solution would be to write a simple script that just looks at the orientation tag, and does the reversal for you. This should be quite simple to do using, for example, pysam.