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

DNA sequencing

Single-read sequencing

• Maxam-Gilbert method

  • Based on chemical degradation

  • NOW OBSOLETE

• Sanger method

  • Based on primer extension chain termination

  • Dominating method until NGS – still used

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

  • Based on primer extension

  • Fully automated

  • A revolution in progress

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

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

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

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

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

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

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)

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

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

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)

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)

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

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

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

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

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