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broadinstitute / infercnv

Inferring CNV from Single-Cell RNA-Seq
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Error in step #15 when running infercnv function #497

Open lunching opened 1 year ago

lunching commented 1 year ago

INFO [2023-01-13 13:01:38] ::process_data:Start INFO [2023-01-13 13:01:38] Checking for saved results. INFO [2023-01-13 13:01:38] 

STEP 1: incoming data

INFO [2023-01-13 13:02:16] 

STEP 02: Removing lowly expressed genes

INFO [2023-01-13 13:02:16] ::above_min_mean_expr_cutoff:Start INFO [2023-01-13 13:02:17] Removing 12480 genes from matrix as below mean expr threshold: 0.1 INFO [2023-01-13 13:02:19] validating infercnv_obj INFO [2023-01-13 13:02:19] There are 5509 genes and 14590 cells remaining in the expr matrix. INFO [2023-01-13 13:02:24] no genes removed due to min cells/gene filter INFO [2023-01-13 13:02:43] 

STEP 03: normalization by sequencing depth

INFO [2023-01-13 13:02:43] normalizing counts matrix by depth INFO [2023-01-13 13:02:45] Computed total sum normalization factor as median libsize: 3279.500000 INFO [2023-01-13 13:02:46] Adding h-spike INFO [2023-01-13 13:02:46] -hspike modeling of SCT1 INFO [2023-01-13 13:03:34] -hspike modeling of SCT2 INFO [2023-01-13 13:04:19] validating infercnv_obj INFO [2023-01-13 13:04:19] normalizing counts matrix by depth INFO [2023-01-13 13:04:19] Using specified normalization factor: 3279.500000 INFO [2023-01-13 13:04:38] 

STEP 04: log transformation of data

INFO [2023-01-13 13:04:38] transforming log2xplus1() INFO [2023-01-13 13:04:40] -mirroring for hspike INFO [2023-01-13 13:04:40] transforming log2xplus1() INFO [2023-01-13 13:05:00] 

STEP 08: removing average of reference data (before smoothing)

INFO [2023-01-13 13:05:00] ::subtract_ref_expr_from_obs:Start inv_log=FALSE, use_bounds=TRUE INFO [2023-01-13 13:05:00] subtracting mean(normal) per gene per cell across all data INFO [2023-01-13 13:05:05] -subtracting expr per gene, use_bounds=TRUE INFO [2023-01-13 13:05:09] -mirroring for hspike INFO [2023-01-13 13:05:09] ::subtract_ref_expr_from_obs:Start inv_log=FALSE, use_bounds=TRUE INFO [2023-01-13 13:05:09] subtracting mean(normal) per gene per cell across all data INFO [2023-01-13 13:05:11] -subtracting expr per gene, use_bounds=TRUE INFO [2023-01-13 13:05:44] 

STEP 09: apply max centered expression threshold: 3

INFO [2023-01-13 13:05:44] ::process_data:setting max centered expr, threshold set to: +/-: 3 INFO [2023-01-13 13:05:46] -mirroring for hspike INFO [2023-01-13 13:05:46] ::process_data:setting max centered expr, threshold set to: +/-: 3 INFO [2023-01-13 13:06:19] 

STEP 10: Smoothing data per cell by chromosome

INFO [2023-01-13 13:06:20] smooth_by_chromosome: chr: chr1 INFO [2023-01-13 13:06:33] smooth_by_chromosome: chr: chr2 INFO [2023-01-13 13:06:46] smooth_by_chromosome: chr: chr3 INFO [2023-01-13 13:06:59] smooth_by_chromosome: chr: chr4 INFO [2023-01-13 13:07:11] smooth_by_chromosome: chr: chr5 INFO [2023-01-13 13:07:23] smooth_by_chromosome: chr: chr6 INFO [2023-01-13 13:07:36] smooth_by_chromosome: chr: chr7 INFO [2023-01-13 13:07:48] smooth_by_chromosome: chr: chr8 INFO [2023-01-13 13:08:01] smooth_by_chromosome: chr: chr9 INFO [2023-01-13 13:08:12] smooth_by_chromosome: chr: chr10 INFO [2023-01-13 13:08:25] smooth_by_chromosome: chr: chr11 INFO [2023-01-13 13:08:37] smooth_by_chromosome: chr: chr12 INFO [2023-01-13 13:08:49] smooth_by_chromosome: chr: chr13 INFO [2023-01-13 13:09:00] smooth_by_chromosome: chr: chr14 INFO [2023-01-13 13:09:12] smooth_by_chromosome: chr: chr15 INFO [2023-01-13 13:09:24] smooth_by_chromosome: chr: chr16 INFO [2023-01-13 13:09:35] smooth_by_chromosome: chr: chr17 INFO [2023-01-13 13:09:47] smooth_by_chromosome: chr: chr18 INFO [2023-01-13 13:09:56] smooth_by_chromosome: chr: chr19 INFO [2023-01-13 13:10:06] smooth_by_chromosome: chr: chr20 INFO [2023-01-13 13:10:16] smooth_by_chromosome: chr: chr21 INFO [2023-01-13 13:10:21] smooth_by_chromosome: chr: chr22 INFO [2023-01-13 13:10:31] -mirroring for hspike INFO [2023-01-13 13:10:31] smooth_by_chromosome: chr: chrA INFO [2023-01-13 13:10:31] smooth_by_chromosome: chr: chr_0 INFO [2023-01-13 13:10:31] smooth_by_chromosome: chr: chr_B INFO [2023-01-13 13:10:32] smooth_by_chromosome: chr: chr_0pt5 INFO [2023-01-13 13:10:32] smooth_by_chromosome: chr: chr_C INFO [2023-01-13 13:10:32] smooth_by_chromosome: chr: chr_1pt5 INFO [2023-01-13 13:10:33] smooth_by_chromosome: chr: chr_D INFO [2023-01-13 13:10:33] smooth_by_chromosome: chr: chr_2pt0 INFO [2023-01-13 13:10:33] smooth_by_chromosome: chr: chr_E INFO [2023-01-13 13:10:33] smooth_by_chromosome: chr: chr_3pt0 INFO [2023-01-13 13:10:34] smooth_by_chromosome: chr: chr_F INFO [2023-01-13 13:11:08] 

STEP 11: re-centering data across chromosome after smoothing

INFO [2023-01-13 13:11:08] ::center_smooth across chromosomes per cell INFO [2023-01-13 13:11:20] -mirroring for hspike INFO [2023-01-13 13:11:20] ::center_smooth across chromosomes per cell INFO [2023-01-13 13:11:57] 

STEP 12: removing average of reference data (after smoothing)

INFO [2023-01-13 13:11:57] ::subtract_ref_expr_from_obs:Start inv_log=FALSE, use_bounds=TRUE INFO [2023-01-13 13:11:57] subtracting mean(normal) per gene per cell across all data INFO [2023-01-13 13:12:01] -subtracting expr per gene, use_bounds=TRUE INFO [2023-01-13 13:12:05] -mirroring for hspike INFO [2023-01-13 13:12:05] ::subtract_ref_expr_from_obs:Start inv_log=FALSE, use_bounds=TRUE INFO [2023-01-13 13:12:05] subtracting mean(normal) per gene per cell across all data INFO [2023-01-13 13:12:07] -subtracting expr per gene, use_bounds=TRUE INFO [2023-01-13 13:12:40] 

STEP 14: invert log2(FC) to FC

INFO [2023-01-13 13:12:40] invert_log2(), computing 2^x INFO [2023-01-13 13:12:41] -mirroring for hspike INFO [2023-01-13 13:12:41] invert_log2(), computing 2^x INFO [2023-01-13 13:13:22] 

STEP 15: Clustering samples (not defining tumor subclusters)

INFO [2023-01-13 13:13:22] define_signif_tumor_subclusters(p_val=0.1 INFO [2023-01-13 13:13:22] define_signif_tumor_subclusters(), tumor: SCT1 INFO [2023-01-13 13:50:48] cut tree into: 1 groups INFO [2023-01-13 13:50:48] -processing SCT1,SCT1_s1 INFO [2023-01-13 13:50:48] define_signif_tumor_subclusters(), tumor: SCT2 INFO [2023-01-13 14:00:00] cut tree into: 1 groups INFO [2023-01-13 14:00:00] -processing SCT2,SCT2_s1 INFO [2023-01-13 14:00:00] -mirroring for hspike INFO [2023-01-13 14:00:00] define_signif_tumor_subclusters(p_val=0.1 INFO [2023-01-13 14:00:00] define_signif_tumor_subclusters(), tumor: spike_tumor_cell_SCT1 INFO [2023-01-13 14:00:01] cut tree into: 1 groups INFO [2023-01-13 14:00:01] -processing spike_tumor_cell_SCT1,spike_tumor_cell_SCT1_s1 INFO [2023-01-13 14:00:01] define_signif_tumor_subclusters(), tumor: spike_tumor_cell_SCT2 INFO [2023-01-13 14:00:01] cut tree into: 1 groups INFO [2023-01-13 14:00:01] -processing spike_tumor_cell_SCT2,spike_tumor_cell_SCT2_s1 INFO [2023-01-13 14:00:01] define_signif_tumor_subclusters(), tumor: simnorm_cell_SCT1 INFO [2023-01-13 14:00:01] cut tree into: 1 groups INFO [2023-01-13 14:00:01] -processing simnorm_cell_SCT1,simnorm_cell_SCT1_s1 INFO [2023-01-13 14:00:01] define_signif_tumor_subclusters(), tumor: simnorm_cell_SCT2 INFO [2023-01-13 14:00:01] cut tree into: 1 groups INFO [2023-01-13 14:00:01] -processing simnorm_cell_SCT2,simnorm_cell_SCT2_s1 INFO [2023-01-13 14:01:22] ::plot_cnv:Start INFO [2023-01-13 14:01:22] ::plot_cnv:Current data dimensions (r,c)=5509,14590 Total=80483683.4599718 Min=0.740718534274863 Max=1.74328021454108. INFO [2023-01-13 14:01:22] ::plot_cnv:Depending on the size of the matrix this may take a moment. INFO [2023-01-13 14:02:26] plot_cnv(): auto thresholding at: (0.865139 , 1.137533) INFO [2023-01-13 14:02:29] plot_cnv_observation:Start INFO [2023-01-13 14:02:29] Observation data size: Cells= 0 Genes= 5509 Error in obs_dendrogram[[1]] : subscript out of bounds In addition: Warning message: In max(nchar(obs_annotations_names)) : no non-missing arguments to max; returning -Inf

GeorgescuC commented 1 year ago

Hi @lunching ,

The last line of the log before the error indicates that there are no cells defined as observations. It seems like all your cells are defined as references since you do have 5509 cells. Infercnv needs some cells as observations for the analysis to be any use.

Regards, Christophe.