Shulou(Shulou.com)06/02 Report--

Today, I will talk to you about how to identify annular RNA in find_circ. Many people may not know much about it. In order to make you understand better, the editor summarized the following content for you. I hope you can get something according to this article.

1. Compare reference genomes

The official pipeline uses bowtie2 software, and the code is as follows

Bowtie2-p16\-- very-sensitive\-- score-min=C,-15,0\-mm\-x hg19-Q\-1 R1.fastq.gz-2 R2.fastq.gz\ 2 > bowtie2.log\ | samtools view-hbuS -\ | samtools sort-accepted_hits

Finally generated a sorted bam file, in fact, this step to choose other comparison software, such as hisat is also possible, only need to generate bam file on it.

two。 Extract a sequence that is not comparable to the reference genome

The unmatched sequences are extracted by samtools software, and the code is as follows

Samtools view-hf 4 accepted_hits.bam | samtools view-Sb-> unmapped.bam3. Extract the anchor sequence from both ends of the sequence

The code is as follows

Unmapped2anchors.py unmapped.bam anchor.fq4. The anchor sequence was compared to the reference genome bowtie2-p 16\-reorder\-mm\-score-min=C,-15,0\-Q-x human_bowtie2_index\-U anchor.fq\-S align.sam5. Forecast circRNA

The code is as follows

Cat align.sam | find_circ.py-G hg19.fa-p hsa_ > splice_sites.bed

The results are as follows

The-p parameter specifies the prefix of the fourth column, and it is recommended to specify the three-letter abbreviation corresponding to the species. It should be noted that both the circular RNA and the linear RNA are included in the sites.bed. The name of the circular RNA is identified by circ, and the name of the linear RNA is identified by norm.

6. Result screening

Filter the results according to the following rules

Filter annular RNA based on keyword CIRCULAR

Removal of cyclic RNA from mitochondria

Filter annular RNA with at least 2 unique junction reads

Removal of annular RNA with unclear fracture point

Filter out the circRNA whose length is greater than 100kb. Here, the 100kb is the genome length, which can be directly subtracted from the head and tail of the circular RNA.

The code is as follows

Grep CIRCULAR splice_sites.bed |\ grep-v chrM |\ awk'$5 > = 2' |\ grep UNAMBIGUOUS_BP |\ grep ANCHOR_UNIQUE |\. / maxlength.py 100000\ > circ_candidates.bed after reading the above, do you have any further understanding of how to identify ring RNA in find_circ? If you want to know more knowledge or related content, please follow the industry information channel, thank you for your support.

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