brownbag-science

Next-Generation Sequencing (NGS)

Just a wee-bit complicated

(Extremely) Generic Steps

1. (Sample) –>
2. Amplified/Template DNA –>
3. Short Reads from Sequencer (FASTQ) –>
4. Mapped reads (BAM) –>
5. sequence !

(figure taken from abmgood.com)

Overall Steps - More Detail

• Sample preparation (extract dna from biological sample)
• Library Preparation, Construction (chop up DNA into fragments)
• Sequencing
  • raw sequencing reads captured: BCL
  • (on sequencer) BCL converted and then converted FASTQ
    • FASTQ = FASTA + quality info
      • Sequence identifier
      • Nucleotide sequence - aka the read
      • Phred quality info per base
• Data Analysis
  • Quality Control, including trimming and/or filtering reads ()
  • Align reads to reference genome: FASTQ –> SAM/BAM
  • Alignment Cleanup: BAM
  • Variant Calling (VCF)
  • Variant Annotation
    • Add information for each variant: symbol, name, transcript, amino acid sequence
    • COSMIC, dbSNP/1000 genomes, panel of normals, etc
  • Variant Filtering - Biological
    • In a clinical setting, usually no matched normal –> Remove unimportant variants
    • Remove known germline variants in population; Improving databases (e.g. dbSNP -> 1000 genomes -> 1000 genomes -> SweGen)
  • Variant Filtering - Technical
    • VAR cutoff, read depth, variant quality score
    • Panel of normals
  • General Quality Control
    • Base quality, GC bias, distinct reads, percent reads mapped, mapping quality, average depth on target region, uniformity, etc

Data Analysis

Steps in a typical next-generation resequencing workflow. De facto standard file formats are given in parentheses.

(figure taken from nature.com)

---

Prev: 05-Medical-Genetics.md

Next: 07-Types-of-NGS.md