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
sed -i 's/\r$//' Iternative_assembly_Pome_Mito.sh
原因解释
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 好像对饮的是 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
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