W1 L3: Sequencing genes and genomes

Sequencing of Biomolecules:

DNA:

  • Work out the order of the four bases (A, C, G, and T) in fragments of DNA, usually amplified by PCR or DNA cloning
  • Possible since 1977 - Increasingly sophisticated and increasingly affordable

RNA:

  • In principle and in practice, RNA is sequenced indirectly through the sequencing of cDNA

Protein:

  • Possible since 1950 (insulin)

Classical protein sequencing is time consuming and requires relatively high amounts →Increasingly replaced by modern methods

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DNA sequencing

Single-read sequencing

  • Maxam-Gilbert method

    • Based on chemical degradation
    • NOW OBSOLETE

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  • Sanger method

    • Based on primer extension chain termination
    • Dominating method until NGS – still used

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  • Massive parallel sequencing = Next-generation sequencing (NGS)

    • Based on primer extension
    • Fully automated
    • A revolution in progress

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Sanger Sequencing

- DNA sequencing with chain-terminating inhibitors

- dideoxy sequencing (ddNPTs)

  • Used for sequencing single genes and fragments of DNA
    • Understanding the structure of the fragment
    • Mutation screening in specific genes
    • Validations of findings from NGS
  • Highly accurate

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Pre-sequencing

  1. Need to produce multiple copies of the DNA fragment to be sequenced

    • Clone the DNA fragment into a plasmid and grow in E Coli or
    • Amplify the DNA fragment by PCR
  2. Denature the sequence by heating (or by adding NaOH) to produce single-stranded DNA

  3. Prepare a DNA polymerase, primer, dNTPs and ddNPTs

Primer Extension

  • DNA synthesis does not start from scratch

  • Primer sequence needed to anneal to template strand

  • Synthesis of new strand - by adding bases to the primer that are complementary to the template→ extending the primer

  • DNA polymerase synthesises complementary strand

    • Starting from the primer
    • Forms a complementary copy to the template strand

Termination of DNA synthesis

Dideoxyribonucleic triphoshates (ddNTPs) — terminator nucleotides

  • Modified version of the normal DNA building blocks (dNTPs)

  • Uses the same bases (A/C/G/T), but the sugar is modified

  • Wherever the ddNTP has been incorporated, DNA synthesis can proceed no further

  • The lack of a 3’ OH group in the dideoxynucleotide prevents the formation of phosphodiester bond

  • Multiple strands due to DNA amplification

  • Excess of normal dNTPs against the amount of ddNTPs (100:1), which compete against each other in DNA synthesis

  • Termination happens at different places at different strands→ result is a set of DNA sequences of varying length, each ending to a ddNTP

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Sanger sequence

  • Primer, DNA polymerase & mix of normal (unlabelled) dNTPs & labeled ddNTPs
  • Labeled used to be radioactive - now fluorescent dyes used
  • Correct order reached through gel electrophoresis & fluorescence detection
  • In genotyping, where only single SNP is genotyped, process is similar, but no normal (unlabelled) dNTPs are added

Separating nucleic acids according to size: Slab gel electrophoresis

  • Nucleid acids cary many -ve charged phosphate gps

  • Migrate towards +ve electrode when placed in electric field

  • Porous gel acts as sieve → small mols. pass more easily than larger ones

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Advances in Sanger sequencing technology

1977-1985

  • Radioactive labelling, requiring 4 separate reaction tubes for each ddNTP - separated individually on large (30cm x 50cm) slab electrophoresis gels→ dry & X-ray
  • X-ray film, heavy X-ray film cassettes, darkroom film manipulation
  • Manual reading of autoradiogram & feeding into computer (re-reading often required)
  • 1.5 days from set up to results

1986 onwards

  • Fluorescent dye labelling - using mixed reaction tube containing all 4 ddNTPs
  • Automated, optical detection system using scanning laser
  • Direct automated entry of DNA base sequence data into computer
  • Time-consuming due to handling of gel plates

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Capillary gel electrophoresis (1995 onwards)

  • Slab gel electrophoresis is manual (slow, prone to human error) - capillary gel electrophoresis is automated
  • Fluorescently-labelled DNA samples migrate through long, v. thin tubes containing polyacrylamide gel (instead of running gel for a finite time)
  • Machine uses laser to detect fluorescence a fixed point just before end of gel
  • Allows longer reads (up to 1000 bases)
    • separation not stopped at specific point
    • each fragment is allowed to proceed to bottom of gel where resolution is highest
  • Faster & cheaper

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Pros & cons of Sanger sequencing

Pros

  • Highly accurate sequences ~99.95%
  • Several 100 bases long (800-1000bp)

Cons

  • Gel eletrophoresis not suitable for handling large no. of samples at a time - not fully automated
    • not suitable to genome sequencing

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1st sequenced human genomes

  • Human Genome Project 2001 → daft genome compromising of DNA from several volunteers
    • BAC - based sequencing (bacterial artificial chromosome)
  • Celera company 2001 → genome of J. Graig Venter
    • Expressed Sequence Tags (ETS)
  • Highly time-consuming & extremely expensive
    • HGP→ took 10 yrs & cost $2.7 billion (In 2022, costs ~$1000)

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Next-generation sequencing (NGS)

  • Also known as massively-parallel sequencing - sequencing millions of DNA fragments simultaneously
  • From 2005 onwards a tech revolution
  • Vast ↑ in amt. of sequencing data per run (seq. throughput) → dramatic ↓in cost
  • Moving from sequencing single gene & exons to:
    • Whole-genome sequences (WGS)
    • Whole-exome sequences (targeted DNA sequencing) (WES)
    • Whole-transcriptomes (RNAseq) & targeted transcriptomes
    • Methyl-Seq, ChIP-seq
  • Ribo-seq

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Key Terminology:

  • Throughput→ amt. of seq. data (in Mb) that are processed in 1 run
  • Read length→ length of DNA fragments -measured in nucleotides
    • short read lengths can be processed w/ high throughput
  • Read depth→ seq. coverage i.e. how many times each seq. is represented
    • 30x to 50x for WGS
    • 100x for WES
    • important for small read lengths for genome assembly
  • Genome assembly→ aligning & merging sequenced pieces to make sense of seq. genome
    • de novo - 1st time sequencing a species or,
    • alignment against a previously obtained reference sequence

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Methods based on amplified DNA templates

  • 2nd-gen DNA seq.
  • From short (35 nucleotides) to medium-length seq. (up to 800 nucleotides)
  • High to v. high seq. throughput
  • Rel. high R of seq. errors in individual reads (overcome by ↑ coverage)

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Commonly used 2nd-gen sequencing platforms

  • Roche/454 pyrosequencing
    • 1st NGS tech in market (2005)- discontinued support in 2016
    • Rel. long reads & speedy but low throughput
    • Emulsion PCR
  • ABI SOLiD technique
    • 2007
    • checks each base independently twice → low error rate
    • “Wildfire” method for preparing seq. templates (v. similar to Bridge PCR by Illumina)
  • Illumina/Solexa sequencing
    • 2008 - now market leader
    • Bridge PCR
  • Ion Torrent systems
    • 2010
    • Emulsion PCR
    • Rel. long reads & speedy but low throughput (similar to Roche/454)

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Sequencing w/ emulsion PCR

  • Uses bead surfaces, H2O & oil

  • Simultaneous amplification of each seq. w/o risk of contamination

  • each bead act as microreactor for PCR - each containing 1 strand of DNA

  • Terminators are reversible - after chem deprotection, synthesis is possible

Sequencing w/ bridge amplification

Whole Process

  • Genomic DNA→ Cut DNA→ Add linkers

    Bridge amplification (𝘪𝘯 𝘴𝘪𝘵𝘶 PCR in figure above)

Methods based on unamplified DNA

  • 3rd-gen DNA sequencing - ‘single-molecule sequencing’ (SMS) → long-read sequencing

  • Long seq. (1000s of nucleotides) -modest throughput

    • important for assembly of genomes from newly seq. species & for distinguishing large-scale variations e.g. copy number variations in known ones
  • Release of protons (instead of fluorophore or phosphate) - recorded as electric current

  • Avoids problems related to DNA amplification (underrepresentation or overrepresentation of DNA after amplification)

  • Simple & cheap tech (small portable machines)

  • Higher error rates

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GTEx portal

  • Genotype-Tissue Expression (GTEx) project - build a comprehensive public resource to study tissue-specific gene expression & regulation
  • 54 non-diseased tissue sites across 1000 individual - for molecular assays inc. WGS, WES & RNA-Seq
  • Remaining samples available from GTEx Biobank
  • GTEx Portal provides open access to data inc. gene expression, QTLs & histology images

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Single-cell sequencing

  • Possible to perform sequencing of genome, transcriptome & epigenome in a single cell
  • Traditional sequencing works on cell pop.→ resulting data are aggregate values
  • For understanding of cell-to-cell variation & identifying new cell types
  • Catalog of human cell types
    • stable cell properties
    • transient cell features
    • cell positions
    • lineage relationships (identification of novel stem cells)
  • Widely employed in cancer research

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Analysis of NGS data

  • Output files consist of millions of short(~100bp) reads (2nd gen) or longer reads (3rd gen)
  • Reads are mapped to reference genome (if species known - as opposed to 𝘥𝘦 𝘯𝘰𝘷𝘰 sequencing)
  • Variants are identified
  • Heavily computational project requiring several software tools & computational skills

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Sanger sequence vs. NGS

  • Sanger sequence
    • cheap, fast, simple
    • highly accurate
    • gives an answer about a single, specific question
  • NGS
    • getting cheaper w/ more tech
    • more error-prone
    • highly versatile (WGS, WES, RNA-Seq, methyl-seq…)
    • computationally laborious

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