BGI data was not suitable for version 2.0.7

[biozhao77]

STAR
--genomeDir ./celescope/reference
--readFilesIn read_2.fq.gz read_1.fq.gz
--readFilesCommand zcat
--soloCBwhitelist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist
--soloCellFilter EmptyDrops_CR 3000 0.99 10 45000 90000 500 0.01 20000 0.001 10000
--outFileNamePrefix .
--runThreadN 10
--clip3pAdapterSeq AAAAAAAAAAAA
--outFilterMatchNmin 50
--soloFeatures Gene GeneFull_Ex50pAS
--outSAMattributes NH HI nM AS CR UR CB UB GX GN
--soloType CB_UMI_Complex
--soloCBposition 0_0_0_7 0_24_0_31 0_48_0_55
--soloUMIposition 0_57_0_68 --soloUMIlen 12
--soloCBmatchWLtype 1MM
--outSAMtype BAM SortedByCoordinate
--soloCellReadStats Standard
--soloBarcodeReadLength 0

When I used 2.0.7 to analysis BGI data (STARsolo step shown up), mapping and counting were finished normally. But, an error "IndexError: sequence length is not enough in R1 read" raised when exec 'celescope.tools.starsolo.get_Q30_cb_UMI'.

I think it was related to the para 'soloCBposition' and 'soloUMIposition ' (the barcode sequence is 68bp, and the library was built with singleron kit v1), but I'm not sure. So I need your help.

Thank you!

[zhouyiqi91]

https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md
singleron kit v1 corresponds to chemistry scopeV2.2.1, which requires at least 69bp R1. If you have 68bp R1, the last 1bp UMI will be thrown away.
change -soloUMIposition 0_57_0_68 --soloUMIlen 12 to -soloUMIposition 0_57_0_67 --soloUMIlen 11 should work.

[zhouyiqi91]

https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md singleron kit v1 corresponds to chemistry scopeV2.2.1, which requires at least 69bp R1. If you have 68bp R1, the last 1bp UMI will be thrown away. change -soloUMIposition 0_57_0_68 --soloUMIlen 12 to -soloUMIposition 0_57_0_67 --soloUMIlen 11 should work.

In multi_rna, use '--chemistry customized --pattern C8L16C8L16C8L1U11T18 --whitelist "./scopeV2.2.1/bclist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist"

[biozhao77]

Thanks for your advice! I found the linker in scopeV2.2.1 was "TCGGTGACAGCCATATCGTAGTCAGAAGCTGAC", and the last base "C" was not sequenced in BGI data. So, the "L1" in '--pattern C8L16C8L16C8L1U11T18' was not exact I think.

[biozhao77]

https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md singleron kit v1 corresponds to chemistry scopeV2.2.1, which requires at least 69bp R1. If you have 68bp R1, the last 1bp UMI will be thrown away. change -soloUMIposition 0_57_0_68 --soloUMIlen 12 to -soloUMIposition 0_57_0_67 --soloUMIlen 11 should work.

In multi_rna, use '--chemistry customized --pattern C8L16C8L16C8L1U11T18 --whitelist "./scopeV2.2.1/bclist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist"

Thanks for your advice! I found the linker in scopeV2.2.1 was "TCGGTGACAGCCATATCGTAGTCAGAAGCTGAC", and the last base "C" was not sequenced in BGI data. So, the "L1" in '--pattern C8L16C8L16C8L1U11T18' was not exact I think.

[zhouyiqi91]

if so，just remove the L1 and use U12

…

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--------------原始邮件-------------- 发件人："biozhao77 ***@***.***>; 发送时间：2024年3月5日(星期二) 晚上9:19 收件人："singleron-RD/CeleScope" ***@***.***>; 抄送："周义其 ***@***.***>;"Comment ***@***.***>; 主题：Re: [singleron-RD/CeleScope] BGI data was not suitable for version 2.0.7 (Issue #276) ----------------------------------- https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md singleron kit v1 corresponds to chemistry scopeV2.2.1, which requires at least 69bp R1. If you have 68bp R1, the last 1bp UMI will be thrown away. change -soloUMIposition 0_57_0_68 --soloUMIlen 12 to -soloUMIposition 0_57_0_67 --soloUMIlen 11 should work. In multi_rna, use '--chemistry customized --pattern C8L16C8L16C8L1U11T18 --whitelist "./scopeV2.2.1/bclist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist" Thanks for your advice! I found the linker in scopeV2.2.1 was "TCGGTGACAGCCATATCGTAGTCAGAAGCTGAC", and the last base "C" was not sequenced in BGI data. So, the "L1" in '--pattern C8L16C8L16C8L1U11T18' was not exact I think. — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you commented.Message ID: ***@***.***>

[biozhao77]

if so，just remove the L1 and use U12
…
---------- 该邮件从移动设备发送
--------------原始邮件-------------- 发件人："biozhao77 @.>; 发送时间：2024年3月5日(星期二) 晚上9:19 收件人："singleron-RD/CeleScope" @.>; 抄送："周义其 @.>;"Comment @.>; 主题：Re: [singleron-RD/CeleScope] BGI data was not suitable for version 2.0.7 (Issue #276) ----------------------------------- https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md singleron kit v1 corresponds to chemistry scopeV2.2.1, which requires at least 69bp R1. If you have 68bp R1, the last 1bp UMI will be thrown away. change -soloUMIposition 0_57_0_68 --soloUMIlen 12 to -soloUMIposition 0_57_0_67 --soloUMIlen 11 should work. In multi_rna, use '--chemistry customized --pattern C8L16C8L16C8L1U11T18 --whitelist "./scopeV2.2.1/bclist ./scopeV2.2.1/bclist ./scopeV2.2.1/bclist" Thanks for your advice! I found the linker in scopeV2.2.1 was "TCGGTGACAGCCATATCGTAGTCAGAAGCTGAC", and the last base "C" was not sequenced in BGI data. So, the "L1" in '--pattern C8L16C8L16C8L1U11T18' was not exact I think. — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you commented.Message ID: @.***>

I'm trying. But I have another question, if the last base "C" will be thrown when L1 was removed? Because I noticed that the linker was 33bp including "C".

[zhouyiqi91]

The lastest pipeline actually does NOT use the sequence of the linker. It just use the --pattern to locate the barcode segments and check if the barcode segments match the whitelist or not.