Mitogenome Retrieval Workflow for LMSE Research
Discover the step-by-step process of retrieving mitogenome data for LMSE by Wenjun Chen. Explore data preparation, assembly strategies, and commands for both reference and de novo approaches. Learn about file organization and script execution for efficient results checking.
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Presentation Transcript
Pipeline of mitogenome retrieving for LMSE Wenjun Chen 2022.1.12
Data preparation Unzip sequence files Separate samples with inline index Remove PCR duplication Remove MALBAC adapter sequences
Strategy for assembly With reference Without reference
With reference Mapping to reference by bwa Commands: reference bwa index ref.fa (make index for reference file) bwa mem ref.fa sample_R1.fq sample_R2.fq > sample.sam (mapping with bwa mem) sequence file mate file generate
With reference Transform *.sam to *.bam and sort by samtools Commands: samtools view bF 4 sample.sam > sample.bam samtools sort sample.bam > sample_sorted.bam
With reference Call consensus by bcftools Commands: bcftools mpileup -Ou -f ref.fa sample_sorted.bam | bcftools call -c | vcfutils.pl vcf2fq | seqtk seq -a > cns.fas
Without reference BLAST the reads to the sequences of conserved regions De novo assemble the reads by Trinity and select contigs Use Novoplasty to extend the contigs
Danio_R1.fq Danio_R2.fq Human_R1.fq Human_R2.fq Danio Human
Files we need to prepare The assembly script: The assembly script: File containing the taxon File containing the taxon name: name: assemble_mito.pl assemble_mito.pl query.txt query.txt Perl module: Perl module: The reference The reference fasta fasta file: file: data.pm data.pm mitoref.fas mitoref.fas Trimmed paired Trimmed paired fastq fastq files: files: taxon_R1.fq taxon_R1.fq taxon_R2.fq taxon_R2.fq
Command perl assemble_mito.pl --query query.txt --reference mitoref.fas
Check the results We used several seeds to start extending, so we need to check if the mitogenome is correct and complete.
Improvements Prepare the sequencing library by using MALBAC directly. Decrease the number of enrichment loci and baits. Make Pacbio or nanopore sequencing library to retrieve long segments DNA.
