What the columns in a BAM file mean
A field-by-field guide to the eleven mandatory SAM/BAM columns, the optional tags that matter, and the commands to read, filter, and visualize your own alignments.
Tag: alignment
11 posts
How to View a BAM File
A practical guide to opening and inspecting BAM alignment files: samtools on the command line, IGV for visual review, and the checks worth running before you trust a variant call.
Genome Mapping Software: What to Use to Align Your Own Genome
A practical guide to the three things people mean by genome mapping, the aligners and flags we would use on a personal whole-genome dataset, and what the compute costs.
Running TopHat for RNA-Seq, and What to Run Instead
A working guide to the TopHat/TopHat2 spliced alignment pipeline: when to use it, the exact commands, and the HISAT2 and salmon pipelines we would use for a personal transcriptome today.
Aligning Your Whole-Genome FASTQs with BWA-MEM
A step-by-step guide to indexing a human reference, running bwa mem on 30x whole-genome FASTQs, and producing a sorted CRAM for variant calling.
How to Align Your Own RNA-Seq Reads to the Human Genome
A working guide to going from FASTQ files to a gene-level count matrix you trust: reference choice, STAR two-pass alignment, Salmon selective alignment, strandedness, and the QC numbers that tell you whether the run is usable.
How to Align RNA-Seq Reads with STAR
A working guide to building a STAR index, running a two-pass spliced alignment of human RNA-seq, and reading the output files that tell you whether the run is any good.
How to Map RNA-seq Reads to the Human Genome
A working guide to aligning and quantifying your own RNA-seq data: reference choice, STAR and Salmon parameters, strandedness, quality thresholds, and the failure modes that silently distort expression estimates.
Whole Genome Mapping: What the Term Means and What It Costs
Genome mapping means at least four different things: historical linkage maps, aligning your reads to a reference, optical genome mapping for structural variants, and the colloquial sense of getting your genome sequenced. Here is how to tell them apart, what each costs, and the pipeline we would run on your own data.
hg19 and GRCh37: what they are, how they differ, and when to still use them
hg19 is UCSC's packaging of the GRCh37 human reference assembly. They are nearly the same sequence with different chromosome names and a different mitochondrion, and the coordinates do not match GRCh38. Here is how to tell which build your files use and how to move between them without corrupting your data.
How to Align and Quantify Your Own RNA-seq Data
A working guide to turning personal RNA sequencing FASTQs into gene- and transcript-level expression estimates: reference choice, selective alignment with Salmon, spliced alignment with STAR, QC thresholds, and the checks that catch a swapped or degraded sample.