作者：donglin204 | 来源：互联网 | 2023-07-25 18:24
1.find_circ的安装
#find_circ需要运行在装有python 2.7的64位系统上，同时需要安装numpy和pysam这两个python模块。其运行需要借助bowtie2和samtools来完成基因组mapping的过程。
wget https://github.com/marvin-jens/find_circ/archive/v1.2.tar.gz
tar -xzvf v1.2.tar.gz
2.参考基因组的下载
#通过fetch_ucsc.py下载ucsc最新版本的参考基因组
fetch_ucsc.py hg19/hg38/mm9/mm10 ref/kg/ens/fa out
3.bowtie2建立参考基因组索引
bowtie2_build hg38.fa hg38
4.基于RNA-Seq的基因组比对（pair-end模式）
###bowtie2参数介绍###
-p 使用多线程；--very-sensitive 允许多重比对，报告出最好的一个；--score-min=C,-15,0 设置比对分数函数；--mm 设置I/O模式。
###samtools view参数介绍###
-h 文件包含header line；-b 输出bam格式；-u 输出非压缩的bam格式 –S 忽略版本兼容
bowtie2 -p 16 --very-sensitive --score-min=C,-15,0 --mm -x /path/to/bowtie2_index -q -1 reads1.fq -2 reads2.fq | samtools view -hbuS - | samtools sort - -o output.bam
5.挑出没有比对上的序列，各取两头20bp短序列（anchor）
samtools view -hf 4 output.bam | samtools view -Sb - > unmapped.bam
python unmapped2anchors.py unmapped.bam | gzip > anchors.qfa.gz
6.根据anchor比对基因组情况寻找潜在的circRNA
bowtie2 -p 16 --reorder --mm -M20 --score-min=C,-15,0 -q -x /path/to/bowtie2_index -U anchors.qfa.gz | python find_circ.py -G /path/to/hg38.fa -p prefix -s find_circ.sites.log > find_circ.sites.bed 2 > find_circ.sites.reads
###根据以下规则对结果进行筛选
1.根据关键词CIRCULAR筛选环状RNA
2.去除线粒体上的环状RNA
3.筛选unique junction reads数至少为2的环状RNA
4.去除断裂点不明确的环状RNA
5.过滤掉长度大于100kb的circRNA,这里的100kb为基因组长度，直接用环状RNA的头尾相减即可
grep CIRCULAR find_circ.sites.bed | grep -v chrM | gawk ‘$5>=2‘ | grep UNAMBIGUOUS_BP | grep ANCHOR_UNIQUE | $path/maxlength.py 100000 > finc_circ.candidates.bed
7.分析多个样本#如果有多个样本，需要分别用find_circ.py运行，然后将各自的结果合并
./merge_bed.py sample1.bed sample2.bed [...] > combined.bed
8.输出的文件格式#前六列为标准的BED文件格式，剩余的12列关于junction的一些信息
| column | name | description |
|---|
1 |
chrom |
chromosome/contig name |
2 |
start |
left splice site (zero-based) |
3 |
end |
right splice site (zero-based).(Always: end > start. 5‘ 3‘ depends on strand) |
4 |
name |
(provisional) running number/name assigned to junction |
5 |
n_reads |
number of reads supporting the junction (BED ‘score‘) |
6 |
strand |
genomic strand (+ or -) |
7 |
n_uniq |
number of distinct read sequences supporting the junction |
8 |
uniq_bridges |
number of reads with both anchors aligning uniquely |
9 |
best_qual_left |
alignment score margin of the best anchor alignment supporting the left splice junction (max=2 * anchor_length) |
10 |
best_qual_right |
same for the right splice site |
11 |
tissues |
comma-separated, alphabetically sorted list of supporting the left splice junction (max=2 * anchor_length) |
12 |
tiss_counts |
comma-separated list of corresponding read-counts |
13 |
edits |
number of mismatches in the anchor extension process |
14 |
anchor_overlap |
number of nucleotides the breakpoint resides within one anchor |
15 |
breakpoints |
number of alternative ways to break the read with flanking GT/AG |
16 |
signal |
flanking dinucleotide splice signal (normally GT/AG) |
17 |
strandmatch |
‘MATCH‘, ‘MISMATCH‘ or ‘NA‘ for non-stranded analysis |
18 |
category |
list of keywords describing the junction. Useful for quick grep filtering |
2019-12-02
22:20:44
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