isovar 1.0.10

isovar

• 概述
• python api
• 命令行
• 内部设计
• 其他isovar命令行工具
• 排序建议

概述

isovar根据癌症rnaseq数据确定突变前后的突变蛋白亚序列。

isovar的工作人员：

1. 收集rna可以读取哪一个跨越了变异的位置，

2. 过滤RNA读到的支持突变的内容，

3. 将突变体读入较长的编码序列，

4. 基于参考注释阅读的突变编码序列匹配 框架，和

5. 将直接由rna决定的编码序列翻译成突变蛋白序列。

组装的编码序列可以包含近端 （生殖系和体细胞）变异，以及任何剪接改变 这是由于修改了拼接信号而导致的。

python api

在下面的示例中，isovar.run_isovar返回isovar.isovarresult对象的列表。 这些对象中的每一个都对应于一个单一的输入变量，并且包含关于该变量所在位置的rna证据以及为该变量组装的任何突变蛋白序列的所有信息。

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam")# this code traverses every variant and prints the number# of RNA reads which support the alt allele for variants# which had a successfully assembled/translated protein sequenceforisovar_resultinisovar_results:# if any protein sequences were assembled from RNA# then the one with most supporting reads can be# accessed from a property called `top_protein_sequence`.ifisovar_result.top_protein_sequenceisnotNone:# print number of distinct fragments supporting the# the variant allele for this mutationprint(isovar_result.variant,isovar_result.num_alt_fragments)

也可以将isovarresult对象的集合展平为pandas数据帧：

fromisovarimportrun_isovar,isovar_results_to_dataframedf=isovar_results_to_dataframe(run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam"))

用于收集rna读取的python api选项

要改变isovar收集和过滤rna读取的方式，可以创建 您自己的isovar.readcollector类的实例，并将其传递给run\isovar

fromisovarimportrun_isovar,ReadCollector# create a custom ReadCollector to change options for how RNA reads are processedread_collector=ReadCollector(use_duplicate_reads=True,use_secondary_alignments=True,use_soft_clipped_bases=True)isovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam",read_collector=read_collector)

用于编码序列汇编和转换的python api选项

要改变isovar如何将rna读入编码序列，需要确定 读取帧和组翻译的氨基酸序列，可以创建 拥有isovar.proteinsequencecreator类的实例并将其传递给run\isovar

fromisovarimportrun_isovar,ProteinSequenceCreator# create a custom ProteinSequenceCreator to change options for how# protein sequences are assembled from RNA readsprotein_sequence_creator=ProteinSequenceCreator(# number of amino acids we're aiming for, coding sequences# might still give us a shorter sequence due to an early stop # codon or poor coverageprotein_sequence_length=30,# minimum number of reads covering each base of the coding sequencemin_variant_sequence_coverage=2,# how much of a reference transcript should a coding sequence match before# we use it to establish a reading framemin_transcript_prefix_length=20,# how many mismatches allowed between coding sequence (before the variant)# and transcript (before the variant location)max_transcript_mismatches=2,# also count mismatches after the variant location toward# max_transcript_mismatchescount_mismatches_after_variant=False,# if more than one protein sequence can be assembled for a variant# then drop any beyond this number max_protein_sequences_per_variant=1,# if set to False then coding sequence will be derived from# a single RNA read with the variant closest to its centervariant_sequence_assembly=True,# how many nucleotides must two reads overlap before they are combined# into a single coding sequencemin_assembly_overlap_size=30)isovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam",protein_sequence_creator=protein_sequence_creator)

用于筛选结果的python api

您可以使用filter\u thresholds选项，通过对象的任何数值属性过滤一个isovarresult对象集合 运行isovar函数。此参数所需的值是一个字典，其键名为'min_fraction_ref_reads'或'max_num_alt_fragments'，其值是数字阈值。 键开头的'min'或'max'之后的所有内容都应该是isovarresult属性的名称。 有关rna读取证据的许多常用属性遵循以下模式：

{num|fraction}_{ref|alt|other}_{reads|fragments} 

例如，在下面g代码过滤结果，使10个或更多的alt读数支持一个变体，并且不超过25%的片段支持ref或alt以外的等位基因。

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam",filter_thresholds={"min_num_alt_reads":10,"max_fraction_other_fragments":0.25})forisovar_resultinisovar_results:# print each variant and whether it passed both filtersprint(isovar_result.variant,isovar_result.passes_all_filters)

未能通过一个或多个筛选器的变体不会从结果集合中排除，但它的相应值中有falsefilter_valuesdictionary属性，并且对于passes_all_filters属性将有一个false值。

如果结果集合展平为数据帧，则每个筛选器都作为列包含。

也可以通过将filter_flags传递到run_isovar来过滤布尔属性（不带数值阈值）。这些布尔值 属性可以通过在属性名前面加上"not_"来进一步否定，以便'protein戋sequence戋u matches戋predicted戋effect'和'not戋protein戋sequence戋u matches戋predicted戋effect'都是筛选标志的有效名称

命令行

基本示例：

$ isovar  \
    --vcf somatic-variants.vcf  \
    --bam rnaseq.bam \
    --protein-sequence-length 30\
    --output isovar-results.csv

加载变量的命令行选项

  --vcf VCF             Genomic variants in VCF format
  
  --maf MAF             Genomic variants in TCGA's MAF format
  
  --variant CHR POS REF ALT
                        Individual variant as 4 arguments giving chromsome,
                        position, ref, and alt. Example: chr1 3848 C G. Use
                        '.' to indicate empty alleles for insertions or
                        deletions.
  
  --genome GENOME       What reference assembly your variant coordinates are
                        using. Examples: 'hg19', 'GRCh38', or 'mm9'. This
                        argument is ignored for MAF files, since each row
                        includes the reference. For VCF files, this is used if
                        specified, and otherwise is guessed from the header.
                        For variants specfied on the commandline with
                        --variant, this option is required.
  
  --download-reference-genome-data
                        Automatically download genome reference data required
                        for annotation using PyEnsembl. Otherwise you must
                        first run 'pyensembl install' for the release/species
                        corresponding to the genome used in your VCF.
  
  --json-variants JSON_VARIANTS
                        Path to Varcode.VariantCollection object serialized as
                        a JSON file.

加载对齐肿瘤rna序列的命令行选项

  --bam BAM             BAM file containing RNAseq reads
  
  --min-mapping-quality MIN_MAPPING_QUALITY
                        Minimum MAPQ value to allow for a read (default 1)
  
  --use-duplicate-reads
                        By default, reads which have been marked as duplicates
                        are excluded.Use this option to include duplicate
                        reads.
                        
  --drop-secondary-alignments
                        By default, secondary alignments are included in
                        reads, use this option to instead only use primary
                        alignments.

用于编码序列程序集的命令行选项

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam")# this code traverses every variant and prints the number# of RNA reads which support the alt allele for variants# which had a successfully assembled/translated protein sequenceforisovar_resultinisovar_results:# if any protein sequences were assembled from RNA# then the one with most supporting reads can be# accessed from a property called `top_protein_sequence`.ifisovar_result.top_protein_sequenceisnotNone:# print number of distinct fragments supporting the# the variant allele for this mutationprint(isovar_result.variant,isovar_result.num_alt_fragments)

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam")# this code traverses every variant and prints the number# of RNA reads which support the alt allele for variants# which had a successfully assembled/translated protein sequenceforisovar_resultinisovar_results:# if any protein sequences were assembled from RNA# then the one with most supporting reads can be# accessed from a property called `top_protein_sequence`.ifisovar_result.top_protein_sequenceisnotNone:# print number of distinct fragments supporting the# the variant allele for this mutationprint(isovar_result.variant,isovar_result.num_alt_fragments)

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam")# this code traverses every variant and prints the number# of RNA reads which support the alt allele for variants# which had a successfully assembled/translated protein sequenceforisovar_resultinisovar_results:# if any protein sequences were assembled from RNA# then the one with most supporting reads can be# accessed from a property called `top_protein_sequence`.ifisovar_result.top_protein_sequenceisnotNone:# print number of distinct fragments supporting the# the variant allele for this mutationprint(isovar_result.variant,isovar_result.num_alt_fragments)

fromisovarimportrun_isovarisovar_results=run_isovar(variants="cancer-mutations.vcf",alignment_file="tumor-rna.bam")# this code traverses every variant and prints the number# of RNA reads which support the alt allele for variants# which had a successfully assembled/translated protein sequenceforisovar_resultinisovar_results:# if any protein sequences were assembled from RNA# then the one with most supporting reads can be# accessed from a property called `top_protein_sequence`.ifisovar_result.top_protein_sequenceisnotNone:# print number of distinct fragments supporting the# the variant allele for this mutationprint(isovar_result.variant,isovar_result.num_alt_fragments)