Kallisto Bus doesn't give output

[rbarbieri86]

Hello,

I am trying to have scUTRquant analyze a small scRNA-seq dataset (3 controls, 3 alternate). I have written the config.yaml and the sample.csv and placed them in a different folder than the scUTRquant one. The files should be correctly pointed at with a full path, however I get this error:

snakemake --use-conda --cores 8 --configfile ~/athero_scRNA/config.yaml
Config file config.yaml is extended by additional config specified via the command line.
[INFO] scUTRquant v0.5.0
[INFO] Loading sample data...
[INFO] Loaded 2 samples.
Assuming unrestricted shared filesystem usage.
Building DAG of jobs...
MissingInputException in rule mtxs_to_sce_genes in file /q/home/barbieri/scUTRquant/Snakefile, line 315:
Missing input files for rule mtxs_to_sce_genes:
    output: data/sce/utrome_mm10_v2/Athero_Clement.genes.Rds
    wildcards: target=utrome_mm10_v2
    affected files:
        data/kallisto/utrome_mm10_v2/A9/genes.barcodes.txt
        data/kallisto/utrome_mm10_v2/A10/genes.mtx
        data/kallisto/utrome_mm10_v2/A9/genes.mtx
        data/kallisto/utrome_mm10_v2/A10/genes.barcodes.txt
        data/kallisto/utrome_mm10_v2/A10/genes.genes.txt
        data/kallisto/utrome_mm10_v2/A9/genes.genes.txt

So it looks like kallisto bus is not providing the expected output. For reference here is the content of the config.yaml I am using:

dataset_name: "Athero_Clement"
sample_file: "/q/home/barbieri/athero_scRNA/Athero_Clement.csv"
sample_regex: ".fastq"
tech: "10xv2"
strand: "--fr-stranded"
cell_annots: null
cell_annots_key: "cell_id"
exclude_unannotated_cells: False
target: "utrome_mm10_v2"
output_type:
  - "genes"
  - "txs"
output_format:
  - "sce"
tmp_dir: "/q/home/barbieri/athero_scRNA/tmp"
bx_whitelist: "/q/home/barbieri/scUTRquant/extdata/bxs/737K-august-2016.txt"
correct_bus: True
min_umis: 500
targets_config: "/q/home/barbieri/scUTRquant/extdata/targets/targets.yaml"
use_hdf5: False
include_reports: True

Many thanks in advance for any pointers :)

[mfansler]

Thanks for your interest and sorry you're having issues getting it going! I'm happy to help resolve the issue.

At first glance, here are some things that come to mind:

• sample_regex is usually not needed; perhaps try removing that
• would you mind also posting the contents of the sample_file so I can confirm that looks good
• have you had any success running one of the examples? that can be a good way to verify everything else is working
• the fact that only the genes output is an issue is unusual; perhaps try leaving that off for now; the txs output is usually the crucial one to get working; worst case, I can show you how to convert from tx- to gene-level output

I apologize in advance if I'm overlooking something. Currently traveling, so I may not be as responsive until Monday.

[mfansler]

Also, the message "Loaded 2 samples." does not match the description of having 2 conditions with 3 replicates. I would expect it should report 6 samples if the sample sheet were correct.

[rbarbieri86]

First of all thanks for your reply, I will try to address your points.

-Ok I will remove the sample_regex -Please look below -Yes I have run both of the suggested examples with success -I can remove the genes part, I am mostly interested in the APA -You might be right, I have obtained the fastq file by using 10x bamtofastq and it does look like the 2 BAM files contained 3 samples each. I will try to restrucure the sample_file accordingly

sample_id,file_type,files A9,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_001.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_001.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_002.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_002.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_003.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_003.fastq A10,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_001.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_001.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_002.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_002.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R1_003.fastq;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/bamtofastq_S1_L007_R2_003.fastq

[rbarbieri86]

Hello, just wanted to report that modifying the sample_file solved the issue. Mine looks like this now:

sample_id,file_type,files A9_1,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_1/A9_1_bamtofastq_S1_L006_R1_001.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_1/A9_1_bamtofastq_S1_L006_R2_001.fastq.gz A9_2,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_2/A9_2_bamtofastq_S1_L006_R1_002.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_2/A9_2_bamtofastq_S1_L006_R2_002.fastq.gz A9_3,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_3/A9_3_bamtofastq_S1_L006_R1_003.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A9/A9_MissingLibrary_1_CB54YACXX/A9_3/A9_3_bamtofastq_S1_L006_R2_003.fastq.gz A10_1,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_1/A10_1_bamtofastq_S1_L007_R1_001.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_1/A10_1_bamtofastq_S1_L007_R2_001.fastq.gz A10_2,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_2/A10_2_bamtofastq_S1_L007_R1_002.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_2/A10_2_bamtofastq_S1_L007_R2_002.fastq.gz A10_3,fastq,/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_3/A10_3_bamtofastq_S1_L007_R1_003.fastq.gz;/q/home/barbieri/athero_scRNA/Fastq_files/A10/A10_MissingLibrary_1_CB54YACXX/A10_3/A10_3_bamtofastq_S1_L007_R2_003.fastq.gz

A quick question: does the sample_id be part of the filenames or is that unnecessary?

Thanks again.

[mfansler]

Great to hear you have it working!

No, the value in sample_id and files column don't need to be coordinated. Consider the sample_id a good opportunity to rename to something more informative.