使用velocyto进行bam转loom吐血踩坑记录

Traceback (most recent call last):
  File "miniconda3/envs/velocyto/bin/velocyto", line 8, in <module>
    sys.exit(cli())
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/click/core.py", line 1137, in __call__
    return self.main(*args, **kwargs)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/click/core.py", line 1062, in main
    rv = self.invoke(ctx)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/click/core.py", line 1668, in invoke
    return _process_result(sub_ctx.command.invoke(sub_ctx))
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/click/core.py", line 1404, in invoke
    return ctx.invoke(self.callback, **ctx.params)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/click/core.py", line 763, in invoke
    return __callback(*args, **kwargs)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/velocyto/commands/run10x.py", line 112, in run10x
    return _run(bamfile=(bamfile, ), gtffile=gtffile, bcfile=bcfile, outputfolder=outputfolder,
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/velocyto/commands/_run.py", line 196, in _run
    mask_ivls_by_chromstrand = exincounter.read_repeats(repmask)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/velocyto/counter.py", line 347, in read_repeats
    gtf_lines = sorted(gtf_lines, key=sorting_key)
  File "miniconda3/envs/velocyto/lib/python3.8/site-packages/velocyto/counter.py", line 345, in sorting_key
    return (x[0], x[6] == "+", int(x[3]), entry)  # The last element of the touple corresponds to the `last resort comparison`
IndexError: list index out of range

2021-09-03 01:07:06,282 - DEBUG - Using logic: Default
2021-09-03 01:07:06,295 - INFO - Read 13734 cell barcodes from Cell_ranger_count/200739E_GDLC_PWZ/outs/filtered_feature_bc_matrix/barcodes.tsv.gz
2021-09-03 01:07:06,296 - DEBUG - Example of barcode: AAACCCAAGATAGTCA and cell_id: 200739E_GDLC_PWZ:AAACCCAAGATAGTCA-1
2021-09-03 01:07:06,306 - WARNING - Your system does not support calling `grep MemAvailable /proc/meminfo` so the memory effort for the samtools command could not be chosen appropriately. 32Gb will be assumed
2021-09-03 01:07:06,306 - DEBUG - Peeking into Cell_ranger_count/200739E_GDLC_PWZ/outs/possorted_genome_bam.bam
2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 10 of the bam file
2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 63 of the bam file
2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 240 of the bam file
2021-09-03 01:07:06,330 - WARNING - Not found cell and umi barcode in entry 544 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 853 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 855 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 860 of the bam file
2021-09-03 01:07:06,336 - WARNING - The file Cell_ranger_count/200739E_GDLC_PWZ/outs/cellsorted_possorted_genome_bam.bam already exists. The sorting step will be skipped and the existing file will be used.
2021-09-03 01:07:06,336 - INFO - Load the annotation from Cell_Ranger/genome/refdata-gex-GRCh38-2020-A/genes/genes.gtf
2021-09-03 01:07:12,783 - DEBUG - Parsing Chromosome GL000009.2 strand - [line 0]
2021-09-03 01:07:12,784 - DEBUG - Done with GL000009.2- [line 7]
2021-09-03 01:07:12,784 - DEBUG - Assigning indexes to genes
2021-09-03 01:07:12,784 - DEBUG - Seen 1 genes until now
2021-09-03 01:07:12,784 - DEBUG - Parsing Chromosome GL000194.1 strand - [line 8]
2021-09-03 01:07:12,784 - DEBUG - Done with GL000194.1- [line 33]
2021-09-03 01:07:12,784 - DEBUG - Assigning indexes to genes
...
...
2021-09-03 01:07:26,663 - DEBUG - Assigning indexes to genes
2021-09-03 01:07:26,664 - DEBUG - Done with Y+ [line 2765967]
2021-09-03 01:07:26,664 - DEBUG - Fixing corner cases of transcript models containg intron longer than 1000Kbp
2021-09-03 01:07:28,588 - DEBUG - Generated 2411536 features corresponding to 199138 transcript models from Cell_Ranger/genome/refdata-gex-GRCh38-2020-A/genes/genes.gtf
2021-09-03 01:07:28,611 - INFO - Load the repeat masking annotation from /share/nas5/zhangj/project/SingleCell_Analysis_Tools/velocyto/hg38_rmsk.gtf
2021-09-03 01:07:28,611 - DEBUG - Reading hg38_rmsk.gtf, the file will be sorted in memory

# 创建环境并激活
conda create -y -n velocyto python=3.8
conda activate velocyto
python --version

# 安装依赖包
conda install -y numpy scipy cython numba matplotlib scikit-learn h5py click
conda install samtools

# 更新 setuptools 和 pip
pip install --upgrade setuptools
python -m pip install --upgrade pip
pip install -i https://pypi.tuna.tsinghua.edu.cn/simple pysam


# 安装velocyto
pip install -i https://pypi.tuna.tsinghua.edu.cn/simple velocyto

# 检查是否成功
velocyto --help
Usage: velocyto [OPTIONS] COMMAND [ARGS]...

Options:
  --version  Show the version and exit.
  --help     Show this message and exit.

Commands:
  run            Runs the velocity analysis outputting a loom file
  run10x         Runs the velocity analysis for a Chromium Sample
  run-dropest    Runs the velocity analysis on DropEst preprocessed data
  run-smartseq2  Runs the velocity analysis on SmartSeq2 data (independent bam file per cell)
  tools          helper tools for velocyto

# 版本
velocyto, version 0.17.17


# 安装4.1.0版本的R
conda install r-base=4.1.0

# 依赖包
conda install -y bioconductor-pcamethods
conda install -y r-velocyto.r

# 先给bam按照CB排序
nohup samtools sort -@ 10  -t CB -O BAM -o cellsorted_possorted_genome_bam.bam possorted_genome_bam.bam &

# 运行
velocyto/bin/velocyto run10x -m hg38_rmsk.gtf   Cell_ranger_count/200739C_GDLC_WXJ Cell_Ranger/genome/refdata-gex-GRCh38-2020-A/genes/genes.gtf

zless -S outs/raw_feature_bc_matrix/features.tsv.gz

2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 10 of the bam file
2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 63 of the bam file
2021-09-03 01:07:06,329 - WARNING - Not found cell and umi barcode in entry 240 of the bam file
2021-09-03 01:07:06,330 - WARNING - Not found cell and umi barcode in entry 544 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 853 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 855 of the bam file
2021-09-03 01:07:06,331 - WARNING - Not found cell and umi barcode in entry 860 of the bam file

2021-09-03 02:42:31,326 - DEBUG - Fixing corner cases of transcript models containg intron longer than 1000Kbp
2021-09-03 02:42:35,990 - DEBUG - Generated 2411536 features corresponding to 199138 transcript models from Cell_Ranger/genome/refdata-gex-GRCh38-2020-A/genes/genes.gtf
2021-09-03 02:42:36,015 - INFO - Load the repeat masking annotation from /share/nas5/zhangj/project/SingleCell_Analysis_Tools/velocyto/GRCh38_rmsk.gtf
2021-09-03 02:42:36,015 - DEBUG - Reading /share/nas5/zhangj/project/SingleCell_Analysis_Tools/velocyto/GRCh38_rmsk.gtf, the file will be sorted in memory
2021-09-03 02:43:17,308 - DEBUG - Processed masked annotation .gtf and generated 4525530 intervals to mask!
2021-09-03 02:43:17,661 - INFO - Scan Cell_ranger_count/200739E_GDLC_PWZ/outs/possorted_genome_bam.bam to validate intron intervals
2021-09-03 02:43:20,862 - DEBUG - Reading Cell_ranger_count/200739E_GDLC_PWZ/outs/possorted_genome_bam.bam
2021-09-03 02:43:20,881 - DEBUG - Read first 0 million reads
2021-09-03 02:43:20,881 - DEBUG - Marking up chromosome 1
2021-09-03 02:45:19,495 - DEBUG - Read first 10 million reads