generate-plot error

[abmmki]

Hi,

I am using 0.23a version. At the generate-plot step it looks like all the required plots I got, however, I see error message at the end. Just wonder if I am missing anything? How to overcome this?

Plotting knee plots There were 16 warnings (use warnings() to see them) Plotting base stats Loading required package: magrittr geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' geom_smooth() using method = 'loess' Error in [<-.data.frame(*tmp*, i, 3, value = c(39858902L, 28250991L : replacement has 2 rows, data has 1 Calls: plotBCDrop -> [<- -> [<-.data.frame Execution halted Traceback (most recent call last): File "/bin/dropSeqPipe", line 9, in load_entry_point('dropSeqPipe==0.23a0', 'console_scripts', 'dropSeqPipe')() File "/usr/lib/python3.4/site-packages/dropSeqPipe/main.py", line 182, in main shell(base_summary) File "/usr/lib/python3.4/site-packages/snakemake-3.10.1-py3.4.egg/snakemake/shell.py", line 80, in new raise sp.CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'Rscript /usr/lib/python3.4/site-packages/dropSeqPipe/Rscripts/singleCell/rna_metrics.R /hd2/' returned non-zero exit status 1

Thanks

[Hoohm]

Hello, sadly the 0.23a version still has a whole Rscript for all of those plots so it's difficult to debug. This will be fixed in 0.24. I guess the main issue is that one of the log files is probably empty. Maybe your filters are too loose. Could you tell me which plot has been produced and which not?

[abmmki]

HI,

I got following 7 plots for each of my 16 samples, and none are empty.

SAMPLE_base_freq_plot.pdf
SAMPLE_Cell_qual.pdf
SAMPLE_knee_plot.pdf
SAMPLE_polya_trimmed.pdf
SAMPLE_rna_metrics.pdf SAMPLE_SMART_trimmed.pdf SAMPLE_UMI_qual.pdf

I also got fastqc.pdf plot. Just which is empty is Rplot.pdf file. .... and I think it is supposed to be empty (?).

best,

[Hoohm]

I see, you might be missing the BCDrop.pdf which is the summary of cell barcode and UMI filtering. Did you run the fastqc step? You must have since you have the plot. Could you please paste the config file?

[abmmki]

Yes, I run the fastqc step first. And got the FastQC plot pdf file.

config.yaml

Samples: S1: fraction: 0.0001 expected_cells: 350 S2: fraction: 0.0001 expected_cells: 400 S3: fraction: 0.0001 expected_cells: 400 S4: fraction: 0.0001 expected_cells: 900 S5: fraction: 0.00005 expected_cells: 1000 S6: fraction: 0.00005 expected_cells: 950 S7: fraction: 0.00002 expected_cells: 2000 S8: fraction: 0.00005 expected_cells: 900 S9: fraction: 0.00005 expected_cells: 1400 S10: fraction: 0.00005 expected_cells: 800 S11: fraction: 0.0001 expected_cells: 1000 S12: fraction: 0.00002 expected_cells: 2000 S13: fraction: 0.00005 expected_cells: 2300 S14: fraction: 0.00002 expected_cells: 1700 S15: fraction: 0.00003 expected_cells: 1800 S16: fraction: 0.00003 expected_cells: 2200 GENOMEREF: /home/bin/DropSeqMetaData/dmel.6.9.fa REFFLAT: /home/bin/DropSeqMetaData/dmel.6.9.refFlat METAREF: /home/bin/DropSeqMetaData/STAR_INDEX_NO_GTF RRNAINTERVALS: /home/bin/DropSeqMetaData/dmel.6.9.rRNA.intervals GTF: /home/bin/DropSeqMetaData/dmel.6.9.gtf SPECIES:

• DROSOPHILA GLOBAL: 5PrimeSmartAdapter: AAGCAGTGGTATCAACGCAGAGT data_type: singleCell allowed_aligner_mismatch: 10 min_count_per_umi: 1 Cell_barcode: start: 1 end: 12 min_quality: 10 num_below_quality: 1 UMI: start: 13 end: 20 min_quality: 10 num_below_quality: 1

[abmmki]

would it be possible that I am missing it as I added following parameter in post_align.snak

VALIDATION_STRINGENCY=LENIENT\