文献笔记二十九：银合欢（Leucaena trichandra）线粒体基因组

文章题目

PacBio-Based Mitochondrial Genome Assembly of Leucaena trichandra (Leguminosae) and an Intrageneric Assessment of Mitochondrial RNA Editing

发表期刊、单位、年份

GBE Genome Biology and Evolution Accepted: August 17, 2018 New Mexico State University Department of Systematic and Evolutionary Botany, University of Zurich, Switzerland（苏黎世大学）论文本地存储名：evy179.pdf

现阶段还是重点关注完整线粒体的组装方法，原文数据公开，还公布了组装使用的shell脚本，争取重复组装过程

DNA Extraction, and Sequencing

sapling 树苗 polysaccharide 多糖 Aquagenomic DNA extraction protocol For each extraction 10 mg of fresh young leaf material was obtained from a L. trichandra sapling that had been kept in the dark for 24h to reduce polysaccharide concentration. DNA with an average fragment size of 21 kbp was submitted for sequencing. PacBIo P6-C4 chemistry

Genome Assembly

followed an iterative approach begins with the assembly of highly conserved regions and extends from that starting point. The pipeline involved:

using BLASR to map raw reads against the reference
filtering hits by a minumum aligned length (500 bp)
recovering the qualifying reads to a new fastq file using seqtk
assembling reads with Canu.

The L.trichandra PacBio reads provided sufficient long read data to also assemble the mitochondrial genome. Nonetheless, when we identified likely mt-genome contigs recovered from assemblies derived from all the available reads (which includes mitochndrial, nuclear, and plastid data in large computationally intensive analyses), the mitochondrial portion was moderately fragmented (> 7 contigs).

计算机资源：The project primarily employed an AMD7252 32 core server with 256 GB of RAM.

将路径改和数据替换为自己的以后运行脚本，遇到报错

[Pomgroup@localhost Pome_Mito_practice]$ bash Iternative_assembly_Pome_Mito.sh 
Iternative_assembly_Pome_Mito.sh: line 2: $'r': command not found
Iternative_assembly_Pome_Mito.sh: line 4: syntax error near unexpected token `$'r''
'ternative_assembly_Pome_Mito.sh: line 4: `

解决办法

https://hacpai.com/article/1488765818607

sed -i 's/r$//' Iternative_assembly_Pome_Mito.sh

原因解释

https://blog.csdn.net/Lnho2015/article/details/51322289

Linux的基础知识还有好多得仔细看！

脚本对应的链接

https://github.com/cdb3ny/Mitochondrial-Genome-Scripts/blob/master/Iternative_assembly_script.sh

脚本中用到的命令逐行解释

首先是blasr比对用法是

blasr nanopore.fastq reference.fasta --nproc 16 > blasr.out

blasr.out 好像对应的是 https://github.com/PacificBiosciences/blasr/wiki/Blasr-Output-Format

这个链接上的 -m为1

操作输出结果blasr.out

awk '{a=$8-$7;print $0,a;}' blastr.out

第8列减去第7列赋值给a并且将a添加到文件的最后一列

awk '{a=$8-$7;print $0,a;}' blastr.out | sort -n -r -k14,14

按照第14列倒叙排列

awk '{a=$8-$7;print $0,a;}' blastr.out | sort -n -r -k14,14 | awk '$14>500'

第14列大于500的行

awk '{a=$8-$7;print $0,a;}' blastr.out | sort -n -r -k14,14 | awk '$14>500' | cut -d ' ' -f1,1

以空格作为分隔符分割然后提取第一列这样就得到了比对长度大于500的fastq的reads的id

grep -F -x -v -f

这行命令是干什么的还不知道

根据id提取序列（fastq）

seqtk subseq nanopore.fasta  ids.txt > aligned.fastq

canu组装

canu -p hehuan -d hehuan-oxford genomeSize=2000k -nanopore-raw aligned.fastq

最后再用canu软件组装的结果作为参考序列重复这个过程，原论文的脚本for i in 1:10相当于是重复了10次这个过程。

好了，这篇文章暂时看到这里了