How to screen mouse sequences (fastq) for membership to the B16-F10 melanoma lineage using bioinformatic pipelines.
Screening mouse sequences (FASTQ files) for membership to the B16-F10 melanoma lineage involves several bioinformatics steps. Here’s a general pipeline you can follow:
First, ensure the quality of your raw sequencing data.
FastQC
, MultiQC
FastQC
on your FASTQ files to generate quality reports.MultiQC
to aggregate the reports for easier interpretation.fastqc sample_R1.fastq sample_R2.fastq
multiqc .
Remove low-quality bases and adapter sequences.
Trimmomatic
, Cutadapt
Trimmomatic
to trim adapters and low-quality bases.trimmomatic PE -phred33 sample_R1.fastq sample_R2.fastq \
sample_R1_paired.fastq sample_R1_unpaired.fastq \
sample_R2_paired.fastq sample_R2_unpaired.fastq \
ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Align the reads to the mouse reference genome.
BWA
, Bowtie2
bwa index GRCm38.fa
bwa mem GRCm38.fa sample_R1_paired.fastq sample_R2_paired.fastq > sample_aligned.sam
Convert the SAM file to BAM format and sort it.
SAMtools
samtools view -Sb sample_aligned.sam > sample_aligned.bam
samtools sort sample_aligned.bam -o sample_sorted.bam
Mark duplicate reads to avoid biases in downstream analysis.
Picard
picard MarkDuplicates I=sample_sorted.bam O=sample_dedup.bam M=sample_metrics.txt
Identify variants that are characteristic of the B16-F10 melanoma lineage.
GATK
, FreeBayes
gatk HaplotypeCaller -R GRCm38.fa -I sample_dedup.bam -O sample_variants.vcf
Filter the called variants to retain high-confidence variants.
GATK
gatk VariantFiltration -R GRCm38.fa -V sample_variants.vcf -O sample_filtered_variants.vcf \
--filter-expression "QD < 2.0 || FS > 60.0 || MQ < 40.0" --filter-name "basic_snp_filter"
Compare the filtered variants with known B16-F10 melanoma lineage-specific variants.
bcftools
, BEDTools
bcftools
or BEDTools
to intersect your variants with the known B16-F10 variants.bcftools isec -p isec_output -n=2 sample_filtered_variants.vcf B16-F10_known_variants.vcf
Analyze the intersection results to determine the membership of your sequences to the B16-F10 melanoma lineage.
Visualize the results for better interpretation.
IGV
, Integrative Genomics Viewer
igv.sh
This pipeline involves quality control, trimming, alignment, duplicate marking, variant calling, filtering, and comparison with known B16-F10 variants. Each step uses specific bioinformatics tools to ensure accurate and reliable results. Adjust the parameters and tools based on your specific requirements and data characteristics.