guide-counter

一种更好的、更快的方法来统计CRISPR筛选中的指南。

概述

guide-counter是一个工具，用于处理CRISPR筛选实验的FASTQ文件，以生成每个样本的指南计数矩阵。它可以作为比mageck count更快、更准确的替代品。默认情况下，guide-counter将在与预期指南相比有0或1个错配的读取中查找指南序列，但也可以以精确匹配模式运行。

为什么选择guide-counter？

如果您有分析CRISPR筛选的经验，您几乎肯定尝试过mageck。它被广泛使用，高度引用，并且通常表现良好。然而，出人意料的是，mageck count既慢又漏掉了相当数量的数据。

例如，我们将来自Sanson等人论文中的数据通过这两个工具运行。数据集包括

以下图表显示了每个样本通过三种不同分析恢复的数据量

以下图表显示了使用单个CPU核心/线程在配备Intel Core i9的MacBook Pro笔记本电脑上执行的三种分析运行时间

安装

可以使用conda进行安装

conda install -c bioconda guide-counter

或者如果有安装，可以使用cargo

cargo install guide-counter

示例工作流程

以下显示了在Sanson等人2018年论文数据上运行guide-counter然后运行mageck test的示例

guide-counter count \
  --input plasmid.fq.gz RepA.fq.gz RepB.fq.gz RepC.fq.gz \
  --control-pattern control \
  --essential-genes metadata/training_essentials.txt \
  --nonessential-genes metadata/training_nonessential.txt \
  --library metadata/broadgpp-brunello-library-corrected.txt.gz  \
  --output sanson
  
mageck test \
  --count-table sanson.counts.txt \
  --control-id plasmid \
  --treatment-id RepA,RepB,RepC \
  --norm-method median \
  --output-prefix sanson.test
  

输入

以下重现了guide-counter count的完整用法；本节详细描述了一些关键输入

输出

输出文件生成

1. {output}.counts.txt - 一个标准计数矩阵，包含引导ID和基因的列，然后是每个样本的原始/未归一化引导计数列。
2. {output}.-extended-counts.txt - 计数矩阵的扩展版本，包含一个guide_type列，其中每个引导将根据提供的基因列表和控制信息确定一个[Essential, Nonessential, Control, Other]。
3. {output}.stats.txt - 一个计算统计文件，每行一个输入样本/FASTQ。

统计文件中的列

使用方法

使用方法：guide-counter count

guide-counter-count

Counts the guides observed in a CRISPR screen, starting from one or more FASTQs.  FASTQs are one per
sample and currently only single-end FASTQ inputs are supported.

A set of sample IDs may be provided using `--samples id1 id2 ..`.  If provided it must have the same
number of values as input FASTQs.  If not provided the FASTQ names are used minus any fastq/fq/gz
suffixes.

Automatically determines the range of valid offsets within the sequencing reads where the guide
sequences are located, independently for each FASTQ input.  The first `offset-sample-size` reads
from each FASTQ are examined to determine the offsets at which guides are found. When processing the
full FASTQ, checks only those offsets that accounted for at least `offset-min-fraction` of the first
`offset-sample-size` reads.

Matching by default allows for one mismatch (and no indels) between the read sub-sequence and the
expected guide sequences.  Exact matching may be enabled by specifying the `--exact-match` option.

Two output files are generated.  The first is named `{output}.counts.txt` and contains columns for
the guide id, the gene targeted by the guide and one count column per input FASTQ with raw/un-
normalized counts.  The second is named `{output}.stats.txt` and contains basic QC statistics per
input FASTQ on the matching process.

USAGE:
    guide-counter count [OPTIONS] --input <INPUT>... --library <LIBRARY> --output <OUTPUT>

OPTIONS:
    -c, --control-guides <CONTROL_GUIDES>
            Optional path to file with list control guide IDs.  IDs should appear one per line and
            are case sensitive

    -C, --control-pattern <CONTROL_PATTERN>
            Optional regular expression pattern used to ID control guides. Pattern is matched, case
            insensitive, to guide IDs and Gene names

    -e, --essential-genes <ESSENTIAL_GENES>
            Optional path to file with list of essential genes.  Gene names should appear one per
            line and are case sensitive

    -f, --offset-min-fraction <OFFSET_MIN_FRACTION>
            After sampling the first `offset_sample_size` reads, use offsets that

            [default: 0.005]

    -h, --help
            Print help information

    -i, --input <INPUT>...
            Input fastq file(s)

    -l, --library <LIBRARY>
            Path to the guide library metadata.  May be a tab- or comma-separated file.  Must have a
            header line, and the first three fields must be (in order): i) the ID of the guide, ii)
            the base sequence of the guide, iii) the gene the guide targets

    -n, --nonessential-genes <NONESSENTIAL_GENES>
            Optional path to file with list of nonessential genes.  Gene names should appear one per
            line and are case sensitive

    -N, --offset-sample-size <OFFSET_SAMPLE_SIZE>
            The number of reads to be examined when determining the offsets at which guides may be
            found in the input reads

            [default: 100000]

    -o, --output <OUTPUT>
            Path prefix to use for all output files

    -s, --samples <SAMPLES>...
            Sample names corresponding to the input fastqs. If provided must be the same length as
            input.  Otherwise will be inferred from input file names

    -x, --exact-match
            Perform exact matching only, don't allow mismatches between reads and guides