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How to handle batch effects in scRNA-seq data using Seurat?
I’m integrating scRNA-seq datasets from 3 different batches (different labs, same tissue type). After merging in Seurat, the UMAP clusters by batch rather than by…
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Fastest way to parse a large VCF file in Python for GWAS analysis?
I have a VCF file with ~15 million SNPs and 5000 samples (~40 GB). I need to extract allele frequencies and filter by MAF >…
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How do I calculate pairwise sequence identity from a multiple sequence alignment in BioPython?
I have a multiple sequence alignment (MSA) in FASTA format and I want to calculate pairwise percent identity for all pairs of sequences. I’m using…
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How do I perform a local BLAST search against a custom protein database in Python?
I have a set of protein sequences in a FASTA file and I want to run a local BLAST search against a custom database I…
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How to analyze 16S rRNA amplicon sequencing data with QIIME2 from raw reads to diversity metrics
I have paired-end 16S V4 amplicon sequencing data (Illumina MiSeq, 250 bp PE reads) from 20 gut microbiome samples. I want to identify taxa, calculate…
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How to use Docker and Singularity to containerize bioinformatics tools for reproducibility
I want to make my bioinformatics analysis fully reproducible using containers. My HPC cluster doesn’t allow Docker (requires root), but Singularity is available. How do…
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What is the best way to normalize RNA-seq count data before differential expression analysis?
I’m doing differential expression analysis with DESeq2 in R. I have raw count data from featureCounts. Should I normalize the counts before passing them to…
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Genome assembly with Flye for long reads: what coverage depth is needed for a good assembly?
I’m assembling a bacterial genome (~4.5 Mb) using Oxford Nanopore reads with Flye. I have about 15x coverage right now. The assembly is fragmented (150+…
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How to annotate protein domains using HMMER hmmscan against the Pfam database
I have 500 novel protein sequences predicted from a de novo genome assembly and I want to annotate them with known functional domains. How do…
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How to build a reproducible bioinformatics pipeline with Snakemake
I want to build a RNA-seq analysis pipeline that I can share with collaborators and rerun on different datasets without manually changing paths. I’ve heard…
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Complete single-cell RNA-seq analysis pipeline in Seurat from CellRanger output to cell type annotation
I have 10x Genomics scRNA-seq data processed through CellRanger. I now have the filtered_feature_bc_matrix output. What is the complete Seurat workflow from loading data to…
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How to parse, filter, and manipulate FASTA files using Biopython
I have a multi-sequence FASTA file with 50,000 protein sequences. I want to: (1) filter sequences by length (keep only 100–500 aa), (2) calculate amino…
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How to create and manage conda environments for reproducible bioinformatics analysis
I keep running into dependency conflicts when installing bioinformatics tools. Different tools require different Python versions and library versions. How should I use conda environments…
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How to perform Gene Set Enrichment Analysis (GSEA) and pathway analysis in R after differential expression
I ran DESeq2 and got a list of differentially expressed genes with log2 fold changes and adjusted p-values. Now I want to understand what biological…