cloneId instead of UMI

[singhd6]

Hi, In the following command of" Load clonesets, lineages and calculate statistics" can I provide the cloneId instead of UMI since I do not have UMIs in my data.
The structure of my ouptput data is:
cloneId readCount readFraction targetSequences targetQualities allVHitsWithScore allDHitsWithScore allJHitsWithScore allCHitsWithScore allVAlignments allDAlignments allJAlignments allCAlignments nSeqFR1 minQualFR1 nSeqCDR1 minQualCDR1 nSeqFR2 minQualFR2 nSeqCDR2 minQualCDR2 nSeqFR3 minQualFR3 nSeqCDR3 minQualCDR3 nSeqFR4 minQualFR4 aaSeqFR1 aaSeqCDR1 aaSeqFR2 aaSeqCDR2 aaSeqFR3 aaSeqCDR3 aaSeqFR4 refPoints
0 4920.0 0.005708491032331872 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCTGGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGGCGATATTGTAGTAGTCCCAGCTGCCCAGTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCAG [[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[ IGHV4-3401(2824) IGHD2-200(101) IGHJ605x(476.4) IGHM00(99.4) 77|371|390|0|294|SG168ASA266TSA267GSG324A|2824.0 33|56|93|294|317|SA47C|101.0 25|83|83|321|379||580.0 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT 58 GGTGGGTCCTTCAGTGATTACTAC 58 TGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAA 58 ATCAATCATAGTGGAAGCACC 58 AACTACAACCCGTCCCTCTGGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCCGCGGACACGGCTGTGTATTAC 58 TGTGCGAGAGGCGATATTGTAGTAGTCCCAGCTGCCCAGTACTACTACTACTACATGGACGTCTGG 58 GGCAAAGGGACCACGGTCACCGTCTCCTCAG 58 QVQLQQWGAGLLKPSETLSLTCAVY GGSFSDYY WSWIRQPPGKGLEWIGE INHSGST NYNPSLWSRVTISVDTSKNQFSLKLNSVTAADTAVYY CARGDIVVVPAAQYYYYYMDVW GKGTTVTVSS_ ::::0:75:99:150:171:282:1:294:294:-2:-6:317:321:-5:348:379::
1 2987.0 0.003465703803572216 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTCTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTAGCTACTACTGGGCCTGGATCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATTGGTGAAATCGATCATAGTGGAAGCACCAACTTCAATCCGTCCCTCGAGAGTCGAGTCTCCATGTCAGTAGACACGTCCAAGAATCAGTTCTACCTGAAGCTGAGGGCTGTGACCGCCGCGGACACGGCTGTTTATTACTGTGCGAGAGGCGGGGGCAGCACCTACTACAGGCCCGGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG [[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[[ IGHV4-3401(2418) IGHD2-200(40),IGHD2-1500(38),IGHD1-2600(35) IGHJ402(370) IGHM00(99.4) 77|371|390|0|294|SG109CSG167AST169CSA179GSG180CSC194TSG223TSA230GSA252TSC256TSA266GSA278TSA283GSC304TSC312ASC325GST326GSG352T|2418.0 16|24|93|305|313||40.0;14|24|93|302|313|I18T|38.0;33|40|60|305|312||35.0 31|68|68|321|358||370.0 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTCTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTAT 58 GGTGGGTCCTTCAGTAGCTACTAC 58 TGGGCCTGGATCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATTGGTGAA 58 ATCGATCATAGTGGAAGCACC 58 AACTTCAATCCGTCCCTCGAGAGTCGAGTCTCCATGTCAGTAGACACGTCCAAGAATCAGTTCTACCTGAAGCTGAGGGCTGTGACCGCCGCGGACACGGCTGTTTATTAC 58 TGTGCGAGAGGCGGGGGCAGCACCTACTACAGGCCCGGTTACTGG 58 GGCCAGGGAACCCTGGTCACCGTCTCCTCAG 58 QVQLQQWGAGLLKPSETLSLTCAVY GGSFSSYY WAWIRQSPGKGLEWIGE IDHSGST NFNPSLESRVSMSVDTSKNQFYLKLRAVTAADTAVYY CARGGGSTYYRPGYW GQGTLVTVSS_ ::::0:75:99:150:171:282:1:294:305:15:-38:313:321:-11:327:358::

Thanks
folder<-"~/webinar/mixcr/"
cloneset_files<-dir(folder,".reassigned_IGH.tsv",full.names = T)

clonesets<-read_tsv(cloneset_files,id="fileName") %>%
mutate(id=str_remove_all(fileName,".\/|.reassigned_IGH.tsv"),
isotype=str_remove(allCHitsWithScore, "\.*"))

stats_clonesets<-clonesets %>%
group_by(id) %>%
summarise(totalCloneInSample=n(),
totalUMIsInSample=sum(uniqueUMICount))

[mizraelson]

You can use readCount instead of UMICount. The Clone ID is simply a unique identifier for the clone, while UMI/Read counts reflect its abundance.

[singhd6]

Hi Mark, Thank you for your reply,

In the below summarise, when I am running the command I have the error:

lng_db<-read_tsv("C:/Users/singhd6/Documents/Lineage_Tree_R/Trees_Data/C2-SUBJ053-IgM_S11_L001_SHMT_Trees.tsv") %>%

• mutate(id=str_remove_all(fileName,".*\/|.reassigned.clns"),

•      isotype=str_remove(bestCHit,"\\*00")) %>% 
  

• separate("id",c("status"),

•        sep="_",
  

•        remove=F) %>%
  

• left_join(stats_clonesets,by ="id")
  Rows: 57131 Columns: 37
  ── Column specification ────────────────────────────────────────────────────────
  Delimiter: "\t"
  chr (27): chains, fileName, targetSequences, targetQualities, bestVHit, best...
  dbl (8): treeId, nodeId, parentId, DistanceFromGermline, nMutationsRate, cl...
  lgl (2): isObserved, allCAlignments

ℹ Use spec() to retrieve the full column specification for this data.
ℹ Specify the column types or set show_col_types = FALSE to quiet this message.
Warning messages:
1: One or more parsing issues, call problems() on your data frame for details,
e.g.:
dat <- vroom(...)
problems(dat)
2: Expected 1 pieces. Additional pieces discarded in 40667 rows [3, 4, 7, 8, 9,
10, 12, 14, 15, 17, 18, 22, 24, 25, 26, 27, 29, 30, 33, 34, ...].

colnames(lng_db)
[1] "treeId" "nodeId" "isObserved"
[4] "chains" "parentId" "DistanceFromGermline"
[7] "nMutationsRate" "cloneId" "fileName"
[10] "readCount" "readFraction" "targetSequences"
[13] "targetQualities" "bestVHit" "bestDHit"
[16] "bestJHit" "bestCHit" "allVAlignments"
[19] "allDAlignments" "allJAlignments" "allCAlignments"
[22] "nSeqFR1" "nSeqCDR1" "nSeqFR2"
[25] "nSeqCDR2" "nSeqFR3" "nSeqCDR3"
[28] "nSeqFR4" "aaSeqFR1" "aaSeqCDR1"
[31] "aaSeqFR2" "aaSeqCDR2" "aaSeqFR3"
[34] "aaSeqCDR3" "aaSeqFR4" "isotype"
[37] "refPoints" "id" "status"
[40] "totalCloneInSample" "totalreadCountInSample"
lngs_trees_wide<-lng_db %>%

• filter(!fileName=="") %>%
• group_by(treeId) %>%
• summarise(nClones=n(),

•         nClonesNormalized=n()/dplyr::first(totalCloneInSample),
  

•         nreadCountNormalized=sum(uniquecloneId)/dplyr::first(totalcloneIdInSample),
  

•         nreadCount=sum(uniquereadCount),
  

•         isotypes=list(unique(isotype)),
  

•         nConditions=n_distinct(status),
  

•         conditions=paste(unique(status),collapse=","),
  

•         totalCloneInSample=dplyr::first(totalCloneInSample),
  

•         totalreadCountInSample=dplyr::first(totalreadCountInSample)
  

• ) %>%
• ungroup() %>%
• group_by(treeId) %>%
• mutate(timepoint=factor(timepoint,levels=c("d0","d21")),

•      timepoints=timepoint %>%unique() %>% sort() %>%  paste(collapse=","),
  

•      isotypes=c(isotypes) %>% unlist() %>% unique() %>%sort() %>%  paste(collapse=","),
  

•      totalNClones=sum(nClones),
  

•      totalCount=sum(readCount)) %>% 
  

• ungroup() %>%
• arrange(timepoint) %>%
• pivot_wider(id_cols = c(treeId,isotypes,timepoints,totalNClones,totalCount),

•           names_from = c(timepoint,repnum),
  

•           values_from = c(nClones,nUMIs,nClonesNormalized,nUMIsNormalized),
  

•           values_fill = 0.00) %>% 
  

• mutate(across(starts_with("nClonesN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x )),

•      across(starts_with("nUmisN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x ))) %>% 
  

• mutate(switchedIsotypePresent=ifelse(str_detect(isotypes, "IGHA|IGHG|IGHE"), TRUE, FALSE))

Error in summarise():
ℹ In argument: nreadCountNormalized = sum(uniquecloneId)/dplyr::first(totalcloneIdInSample).
ℹ In group 1: treeId = 1.
Caused by error:
! object 'uniquecloneId' not found
Run rlang::last_trace() to see where the error occurred.

rlang::last_trace()
<error/rlang_error>
Error in summarise():
ℹ In argument: nreadCountNormalized = sum(uniquecloneId)/dplyr::first(totalcloneIdInSample).
ℹ In group 1: treeId = 1.
Caused by error:
! object 'uniquecloneId' not found

---

Backtrace:
▆

1. ├─... %>% ...
2. ├─dplyr::mutate(...)
3. ├─dplyr::mutate(...)
4. ├─tidyr::pivot_wider(...)
5. ├─dplyr::arrange(., timepoint)
6. ├─dplyr::ungroup(.)
7. ├─dplyr::mutate(...)
8. ├─dplyr::group_by(., treeId)
9. ├─dplyr::ungroup(.)
10. ├─dplyr::summarise(...)
11. └─dplyr:::summarise.grouped_df(...)
12. └─dplyr:::summarise_cols(.data, dplyr_quosures(...), by, "summarise")

13. ├─base::withCallingHandlers(...)
    

14. └─dplyr:::map(quosures, summarise_eval_one, mask = mask)
    

15.   └─base::lapply(.x, .f, ...)
    

16.     └─dplyr (local) FUN(X[[i]], ...)
    

17.       └─mask$eval_all_summarise(quo)
    

18.         └─dplyr (local) eval()
    

Run rlang::last_trace(drop = FALSE) to see 4 hidden frames.

I am thinking what should I provide instead of UMIs in the command: nUMIsNormalized=sum(uniqueUMICount)/dplyr::first(totalUMIsInSample),
nUMIs=sum(uniqueUMICount),
lngs_trees_wide<-lng_db %>%
filter(!fileName=="") %>%
group_by(treeId,timepoint,repnum) %>%
summarise(nClones=n(),
nClonesNormalized=n()/dplyr::first(totalCloneInSample),
nUMIsNormalized=sum(uniqueUMICount)/dplyr::first(totalUMIsInSample),
nUMIs=sum(uniqueUMICount),
isotypes=list(unique(isotype)),
nConditions=n_distinct(status),
conditions=paste(unique(status),collapse=","),
nTissues=n_distinct(tissue),
tissues=paste(unique(tissue),collapse=","),
totalCloneInSample=dplyr::first(totalCloneInSample),
totalUMIsInSample=dplyr::first(totalUMIsInSample)
) %>%
ungroup() %>%
group_by(treeId) %>%
mutate(timepoint=factor(timepoint,levels=c("T1","T2","T3","T4","T5","T6","T7","T8")),
timepoints=timepoint %>%unique() %>% sort() %>% paste(collapse=","),
isotypes=c(isotypes) %>% unlist() %>% unique() %>%sort() %>% paste(collapse=","),
totalNClones=sum(nClones),
totalCount=sum(nUMIs)) %>%
ungroup() %>%
arrange(timepoint) %>%
pivot_wider(id_cols = c(treeId,isotypes,timepoints,totalNClones,totalCount),
names_from = c(timepoint,repnum),
values_from = c(nClones,nUMIs,nClonesNormalized,nUMIsNormalized),
values_fill = 0.00) %>%
mutate(across(starts_with("nClonesN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x )),
across(starts_with("nUmisN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x ))) %>%
mutate(switchedIsotypePresent=ifelse(str_detect(isotypes, "IGHA|IGHG|IGHE"), TRUE, FALSE))

Thank you so much in advance.
Divya

[mizraelson]

What is uniquecloneId and where does it come from?

[singhd6]

Thank you so much Mark,
I specified the "cloneID" form lng_db column names as in:
colnames(lng_db)
[1] "treeId" "nodeId" "isObserved"
[4] "chains" "parentId" "DistanceFromGermline"
[7] "nMutationsRate" "cloneId" "fileName"
[10] "readCount" "readFraction" "targetSequences"
[13] "targetQualities" "bestVHit" "bestDHit"
[16] "bestJHit" "bestCHit" "allVAlignments"
[19] "allDAlignments" "allJAlignments" "allCAlignments"
[22] "nSeqFR1" "nSeqCDR1" "nSeqFR2"
[25] "nSeqCDR2" "nSeqFR3" "nSeqCDR3"
[28] "nSeqFR4" "aaSeqFR1" "aaSeqCDR1"
[31] "aaSeqFR2" "aaSeqCDR2" "aaSeqFR3"
[34] "aaSeqCDR3" "aaSeqFR4" "isotype"
[37] "refPoints" "id" "status"
[40] "totalCloneInSample" "totalreadCountInSample"
What should I provide if I do not have UMIs in my data?

My R script I ran is attached.
[Lineage_Tree.txt](https://github.com/user-attachments/files/18576876/Lineage_Tree.txt)

I am trying to utilize Mixcr's below command to darw lineage abundance scatter plot:
lngs_trees_wide<-lng_db %>%
filter(!fileName=="") %>%
group_by(treeId,timepoint,repnum) %>%
summarise(nClones=n(),
nClonesNormalized=n()/dplyr::first(totalCloneInSample),
nUMIsNormalized=sum(uniqueUMICount)/dplyr::first(totalUMIsInSample),
nUMIs=sum(uniqueUMICount),
isotypes=list(unique(isotype)),
nConditions=n_distinct(status),
conditions=paste(unique(status),collapse=","),
nTissues=n_distinct(tissue),
tissues=paste(unique(tissue),collapse=","),
totalCloneInSample=dplyr::first(totalCloneInSample),
totalUMIsInSample=dplyr::first(totalUMIsInSample)
) %>%
ungroup() %>%
group_by(treeId) %>%
mutate(timepoint=factor(timepoint,levels=c("T1","T2","T3","T4","T5","T6","T7","T8")),
timepoints=timepoint %>%unique() %>% sort() %>% paste(collapse=","),
isotypes=c(isotypes) %>% unlist() %>% unique() %>%sort() %>% paste(collapse=","),
totalNClones=sum(nClones),
totalCount=sum(nUMIs)) %>%
ungroup() %>%
arrange(timepoint) %>%
pivot_wider(id_cols = c(treeId,isotypes,timepoints,totalNClones,totalCount),
names_from = c(timepoint,repnum),
values_from = c(nClones,nUMIs,nClonesNormalized,nUMIsNormalized),
values_fill = 0.00) %>%
mutate(across(starts_with("nClonesN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x )),
across(starts_with("nUmisN"),.fns = ~ ifelse(.x==0, min(.x[.x>0])/2, .x ))) %>%
mutate(switchedIsotypePresent=ifelse(str_detect(isotypes, "IGHA|IGHG|IGHE"), TRUE, FALSE))

[mizraelson]

You can use readCount (not cloneId) instead of UMICount.