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基因组组装 -- pacbio序列组装

一、实验目的

二、知识回顾

三、上机操作

# 登录服务器
# 设置环境变量
module bioinfo
# 准备工作目录和数据
mkdir lab1_2
cd lab1_2
ln -s /bs1/data/genomeLab/lab1.2/data/selfSampleData/pacbio_filtered.fastq ./

一步法

By default, canu will correct the reads, then trim the reads, then assemble the reads to unitigs.

canu \
 -p ecoli -d ecoli-auto \
 genomeSize=4.8m \
 -pacbio-raw pacbio_filtered.fastq

# 组装结果
ecoli-auto/ecoli.contigs.fasta

分步法

第一步：纠错
canu -correct \
  -p ecoli -d ecoli \
  genomeSize=4.8m \
  -pacbio-raw pacbio_filtered.fastq
第二步：trim reads
canu -trim \
  -p ecoli -d ecoli \
  genomeSize=4.8m \
  -pacbio-corrected ecoli/correction/ecoli.correctedReads.fasta.gz
第三步：组装
canu -assemble \
  -p ecoli -d ecoli-erate-0.013 \
  genomeSize=4.8m \
  errorRate=0.013 \
  -pacbio-corrected ecoli/trimming/ecoli.trimmedReads.fasta.gz

While Canu corrects sequences and has 99% identity or greater with PacBio or Nanopore sequences, for the best accuracy we recommend polishing with a sequence-specific tool. We recommend Quiver for PacBio and Nanopolish for Oxford Nanpore data.

If you have Illumina sequences available, Pilon can also be used to polish either PacBio or Oxford Nanopore assemblies.

四、作业与思考

  1. 用miniasm组装同样的数据
  2. 比较miniasm和canu组装的结果

miniasm组装

# Overlap
minimap -Sw5 -L100 -m0 -t8 pacbio_filtered.fastq pacbio_filtered.fastq | gzip -1 > reads.paf.gz
# Layout
miniasm -f pacbio_filtered.fastq reads.paf.gz > reads.gfa
# 将gfa转换为fasta
awk '/^S/{print ">"$2"\n"$3}' reads.gfa | fold > miniasm.fa

五、参考文献

  1. http://canu.readthedocs.io/en/latest/index.html
  2. https://github.com/PacificBiosciences/GenomicConsensus
  3. Koren S, Walenz BP, Berlin K, Miller JR, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. bioRxiv. (2016).