靶向基因表达技术主要针对在测序前直接从单细胞RNA-seq库中富集的相关靶向基因。通过杂交捕获特异性富集感兴趣的靶向分子，使靶向基因表达能够在显著降低测序深度的情况下提供关于靶向基因表达的增强信息。靶向基因表达与Chromium单细胞3’基因表达和5’基因表达解决方案(包括特征条形码技术)完全兼容，并依赖于相同的底层Cell Ranger分析软件。
Cell Ranger使用cellranger count 管道分析单细胞靶向基因表达数据。Cell Ranger为靶向和全转录组分析(WTA)基因表达数据提供了相同的接口和特征，如Single-Library Analysis with Cell Ranger分析所述。
要启用cellranger count中的靶向基因表达分析，请使用Target - Panel标记指定相关的靶向Panel CSV文件:
$ cd /home/jdoe/runs
$ cellranger count --id=sample345 \
--target-panel=/opt/cellranger-4.0.0/target_panels/immunology_v1.0_GRCh38-2020-A.target_panel.csv \
--transcriptome=/opt/refdata-gex-GRCh38-2020-A \
--fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \
--sample=mysample \
--expect-cells=1000 \
--localcores=8 \
--localmem=64
靶向Panel CSV文件
与预先设计的基因Panel 对应的靶向Panel CSV文件可以在Cell Ranger包的 target_panels目录中找到。例如，如果您将cellRanger安装到/opt/cellranger-4.0.0，那么靶向Panel CSVs将包含在/opt/cellranger-4.0.0/target_panels中。这些CSV文件也可以在靶向Panel 选择页面上下载以及关于每个靶向Panel 的更详细的信息。
在这里可以找到关于此文件格式的详细文档，以及为靶向Panel 提供的所有其他下载的描述。
Panel 下载页面
在Panel 选择概述中，我们为所有预先设计的靶基因Panel 提供了包含以下文件的页面链接:
| File | Description |
|---|---|
Gene Metadata File |
A TSV file that lists all the genes included in the final target gene panel. |
Target Panel CSV File |
This CSV file is a required input for Cell Ranger to enable analysis of targeted GEX data. It specifies details of the target genes and bait sequences in the target gene panel. |
Bait BED File |
A BED12 file containing containing the sequences and genomic coordinates for all baits in the target gene panel. Use this file to visualize the bait locations on genome browsers like IGV (Integrated Genomics Viewer) and the UCSC Genome Browser. |
通过10x Genomics定制Panel 设计师创建的定制设计将包括上述每个文件，以及以下附加文件:
| File | Description |
|---|---|
| Bait Design File | Use this Excel (.xlsx) file to order your custom or add-on panel from a compatible oligo provider. It contains the bait sequences for add-on genes and custom sequences. |
| Custom Sequence GTF File | A GTF file corresponding to the custom input sequences entered for panel design. Use this file to create a custom reference compatible with Cell Ranger by appending its contents to the transcriptome GTF file. |
| Custom Sequence FASTA File | A FASTA file corresponding to the custom input sequences entered for panel design. Use this file to create a custom reference compatible with Cell Ranger by appending its contents to the reference FASTA file. |
Note About Bait Identifiers
包含有关单个Bait 信息的文件有一个Bait 标识符(ID)对应的列，该标识符惟一地标识每个Bait ，对于根据需要在不同文件之间匹配条目非常有用。Bait id采用以下格式:
gene_id|gene_name|bait_number
例如，为基因TSPAN6列出的第一个诱饵，其Ensembl ID在GRCh38-2020-A引用中为ENSG00000000003，Bait ID为:
ENSG00000000003|TSPAN6|1
对于自定义序列，Bait ID中的gene_id和gene_name字段将是相同的，并与为每个序列提供的名称匹配。
下载的Panel 格式
这个CSV文件是cellRanger分析目标GEX数据所需的输入。它指定了构成目标基因Panel 的目标基因和Bait 序列。请参阅cellranger count文档中对cellranger count的描述。靶向panel格式：
#panel_name=Human Gene Signature Panel
#panel_type=predesigned
#reference_genome=GRCh38
#reference_version=2020-A
#target_panel_file_format=1.0
gene_id,bait_seq,bait_id
ENSG00000000003,AGTTG[...]GCGTC,ENSG00000000003|TSPAN6|1
ENSG00000000003,CCCGT[...]GGCAA,ENSG00000000003|TSPAN6|2
ENSG00000000003,GGTGA[...]ACCTG,ENSG00000000003|TSPAN6|3
[ ... ]
| Column Name | Description |
|---|---|
| gene_id | The Ensembl gene identifier associated with this bait. |
| bait_seq | The nucleotide sequence of the bait. |
| bait_id | The bait ID associated with this bait formatted as described above. |
元字段信息。
| Metadata Field | Description |
|---|---|
| panel_name | This is the name of the panel. |
| panel_type | Indicates whether this was a predesigned or custom panel. One of predesigned, custom_, or fully_custom. |
| reference_genome | The genome build of cellranger reference baits were designed against. |
| reference_version | The version of the cellranger reference baits were designed against. |
| target_panel_file_format | The version of the target_panel file format specification this file conforms to. |
一般来说，我们强烈建议使用我们提供的目标panel CSV文件下载，或者使用10x Genomics定制panel设计生成的CSV文件。如果确实需要创建自己的目标panel，则必须为所有元数据字段提供条目。gene_id列是必需的，而bait_seq和bait_id列是强烈建议要有的，但却是可选的。bait_id值不需要遵循上面列出的格式，但是它们对每个bait都必须是唯一的。
Bait BED File
bed12格式的文件(下面详细介绍了12列)文件，包含目标基因panel中所有bait的序列和基因组坐标。使用此文件可以在基因组浏览器(如IGV (Integrated Genomics Viewer)和UCSC基因组浏览器)上可视化bait位置，或者执行自定义分析。
chrX 100636685 100636805 ENSG00000000003|TSPAN6|1 0 - 100636685 100636694 0 1 120 0
chrX 100635704 100636685 ENSG00000000003|TSPAN6|2 0 - 100635704 100636685 0 2 42,78 0,903
chrX 100635584 100635704 ENSG00000000003|TSPAN6|3 0 - 100635584 100635704 0 1 120 0
| Column Name | Description |
|---|---|
| chromosome | Chromosome of the target gene. |
| chromStart | 0-based start coordinate of the targeted sequence on the chromosome. |
| chromEnd | 0-based non-inclusive end coordinate on the chromosome. |
| name | Bait ID as described above. |
| score | Set to 0 for all entries. |
| strand | + or - to indicate the strand of the targeted gene. |
| thickStart | The starting position at which the feature is drawn as a thick line in browsers (matches display of the corresponding transcript region). |
| thickEnd | The ending position at which the feature is drawn as a thick line in browsers (matches display of the corresponding transcript region). |
| itemRgb | Set to 0 for all entries. |
| blockCount | The number of blocks (continuous intervals). |
| blockSizes | Comma-separated list of the block sizes, contains blockCount entries. |
| blockStarts | Comma-separated list of block starts relative to chromStart column, contains blockCount entries. |
选择BED12格式是因为它可以方便地将跨越拼接连接点的baits 表示在一条直线上，并允许基因组浏览器将基因组空间中不连续的baits 区域之间的链接可视化。像UCSC Genome Browser或IGV这样的浏览器会适当地呈现BED12文件，以一种与显示基因组中的转录本相似的方式显示。
命令行工具也很好地支持这种格式。例如，bedtools为一些子命令提供了一个-split命令行标志，允许根据需要独立处理BED12文件每行中的各个块。这在计算交点时很有用，例如，您可能对baits 本身覆盖的区域的交叉点感兴趣，而不是对baits 坐标跨度包括内含子区域在内的整个基因组区间的交叉点感兴趣。bedtools还提供了子命令bed12tobed6，用于将BED12文件转换为BED6格式——在生成的文件中，当跨越一个或多个接合点时，每个baits 将出现在多行上。
输出文件
靶基因表达分析的输出通常与非靶基因分析的输出非常相似——详见下表的差异列表。主要的变化是增加了靶向指标，并且所有针对目标数据的二级分析都是在从计数矩阵中去除非目标基因之后进行的。同样，过滤后的特征barcode矩阵文件和Loupe浏览器文件也不包括非目标基因计数。原始的特征barcode矩阵和分子信息H5文件仍然提供所有基因的信息。
| File Name | Description | Differences for Targeted Gene Expression |
|---|---|---|
web_summary.html |
Run summary metrics and charts in HTML format | Contains additional Targeted Gene Expression metrics |
metrics_summary.csv |
Run summary metrics in CSV format | Contains additional Targeted Gene Expression metrics |
possorted_genome_bam.bam |
Reads aligned to the genome and transcriptome annotated with barcode information | The xf tag now indicates whether a read was filtered by targeted UMI filtering |
possorted_genome_bam.bam.bai |
Index for possorted_genome_bam.bam
|
|
filtered_feature_bc_matrix |
Filtered feature-barcode matrices containing only cellular barcodes in MEX format | Contains only targeted genes |
filtered_feature_bc_matrix_h5.h5 |
Filtered feature-barcode matrices containing only cellular barcodes in HDF5 format | Contains only targeted genes* |
raw_feature_bc_matrices |
Unfiltered feature-barcode matrices containing all barcodes in MEX format | |
raw_feature_bc_matrix_h5.h5 |
Unfiltered feature-barcode matrices containing all barcodes in HDF5 format | Generally unchanged* |
analysis |
Secondary analysis data including dimensionality reduction, cell clustering, and differential expression | Secondary analysis performed only on the targeted genes |
molecule_info.h5 |
Molecule-level information used by <samp>cellranger aggr</samp> to aggregate samples into larger datasets. | Generally unchanged* |
cloupe.cloupe |
Loupe Browser visualization and analysis file | Contains only targeted genes |
target_panel.csv |
Target Panel CSV file | Only present for Targeted Gene Expression data |
feature_reference.csv |
(Feature Barcode Only) Feature Reference CSV file |
为了让大家了解一下输出文件我们来看一个标准输出：
我们看一个websummary文件：
Targeted Gene Expression
Target Panel CSV file: A CSV file declaring the target gene panel used for a Targeted Gene Expression experiment, which specifies detailed information about the targeted genes and baits included in the panel. This file must be provided to <samp>cellranger count</samp> via the --target-panel option when analyzing Targeted Gene Expression data. Details and specifications of the Target Panel CSV file format are documented here.
Baits: A set of oligonucleotide sequences designed to specifically hybridize to and recover molecules from targeted genes during the hybridization-capture enrichment step of the Targeted Gene Expression assay.
WTA (or Whole Transcriptome Analysis): Whole Transcriptome Analysis, describes a (non-targeted) Chromium single-cell RNA-seq library or dataset which may be used as input for a Targeted Gene Expression experiment.
Parent: A WTA single-cell gene expression library or dataset that was used as the input material for a Targeted Gene Expression experiment. Because the Parent and its corresponding Targeted dataset are both derived from the same Library and/or GEM well, it is possible to directly compare results from these matched datasets in a pairwise manner, as enabled by <samp>cellranger targeted-compare</samp>.
