○ Meaning of determining restriction enzyme recognition sites: Using the information of fragments with known sequences based on each restriction enzyme recognition site to cumulatively construct a physical map

○ Meaning of constructing a gene map: Once a physical map is constructed, it can be compared with a gene map. Introns exist between genes

○ Approach using a single library

⑶ Methodology 2: Shotgun Sequencing (Celera, J. Craig Venter) (Entrepreneurial Approach)

① Cutting one DNA in multiple ways

② Determining the sequence of fragments cut by one method

○ The length of the analysis sample is limited, so the DNA sequence cannot be determined all at once

③ Randomly arranging the sequences of bases in each method until a common result is obtained

④ Method based on computer science

⑷ Scientists’ Approach vs. Entrepreneurial Approach

① Scientists’ approach is dissatisfied with entrepreneurs’ appropriation of their contributions and investments

② Entrepreneurial approach is dissatisfied with scientists’ failure to disclose information, leading to the development of new methodologies

③ The final completion of the genome map is a joint effort of both.

Figure 1. Stepwise Sequencing Method and Shotgun Sequencing Method

2. Sequencing Technology

⑴ Overview

① DNA Sequencing: Applying the principle of DNA replication

○ Template: Each strand of DNA

○ Substrate: dNTP (dATP, dCTP, dGTP, dTTP)

○ NTP has an -OH group at the 2’ carbon and is a material for RNA synthesis

○ DNA polymerase: Phosphate of the next nucleotide binds to the 3’-OH of deoxyribose

○ Synthesis direction: 5’ → 3’, forming complementary base pairs with the template

○ ddNTP: DNA polymerization is terminated because the 3^rd carbon lacks an OH group

② RNA Sequencing: Applying the principle of RNA transcription

⑵ in vitro cloning: The very first sequencing method

⑶ Dideoxy Chain Termination Method (= Sanger sequencing): Reported in 1977, Sanger’s second Nobel Prize

① Substrate: dNTP + ddNTP (in small quantities) + buffer (pH stabilized)

○ ddNTP lacks a 3’-OH group, so it terminates the polymerization reaction

○ If ddNTP is added in large quantities, all template DNAs are quickly terminated

② Primer

○ Example: p32-primer (CTAG)

③ 1^st. Addition of template DNA and polymerase

④ 2^nd. Heating to separate the complementary strand after polymerization

⑤ 3^rd. Electrophoresis followed by reading the sequence on X-ray film or fluorescence examination

Figure 2. Process of Dideoxy Chain Termination Method

⑥ Advantages: Can read very long strands, still used in laboratories

⑦ Disadvantages: Requires a large amount of the same DNA strand

⑷ Dye-dideoxy chain termination method : Using laser

① Add a small amount of ddNTP to 4-color fluorescent dNTP.

② Automatic DNA sequencing is possible.

Figure 3. Process of the Dye-dideoxy chain termination method

⑸ Pyrosequencing

① Definition : A DNA sequencing method that relies on the proportional luminescence produced based on the amount of pyrophosphate generated during DNA synthesis.

② Diagram

Figure 4. Pyrosequencing diagram

③ Process

Figure 5. Pyrosequencing process

⑹ Illumina solid-phase amplification (ref)

Figure 6. Illumina solid-phase amplification

Figure 7. Fluorescent color distribution photo

① 1^st. Fragmentation : Randomly cut the given DNA sample.

② 2^nd. Gel-based size selection : Size of each DNA fragment can be limited if necessary.

③ 3^rd. Adaptor binding : Attach an adapter to both ends of all DNA sample fragments.

④ 4^th. Amplification

○ 4^th - 1^st. Denature DNA into single-strands.

○ 4^th - 2^nd. Attach single-stranded DNA to the Illumina flow cell.

○ 4^th - 3^rd. Add enzymes to allow single-stranded DNA to form bridges on a solid-phase substrate.

○ 4^th - 4^th. After adding primers to the single-stranded DNA bridges, primers can bind to the bridges.

○ 4^th - 5^th. Add unlabeled single-stranded DNA and induce DNA synthesis : Forms double-stranded DNA bridges.

○ 4^th - 6^th. Denature to turn double-stranded DNA bridges into anchored single-stranded DNA.

○ 4^th - 7^th. Repeat the above six steps to create anchored single-stranded clusters with the same base sequence.

○ Feature **: Anchored single-stranded clusters form millions of clusters.

⑤ 5^th. Sequencing by synthesis (SBS)

○ 4^th - 1^st. Add 4 types of labeled reversible terminators, primers, and DNA polymerase according to the base type.

○ 4^th - 2^nd. When labeled reversible terminators form phosphodiester bonds, fluorescence is emitted.

○ 4^th - 3^rd. Obtain a fluorescent color distribution image of each cluster.

○ 4^th - 4^th. Washing

○ 4^th - 5^th. Repeat the above four steps to determine the entire base sequence.

○ Type 1. Single-end sequencing (SES) : Sequencing with only one adapter.

○ Type 2. Paired-end sequencing (PES) : Sequencing with both adapters.

○ Initially, sequence with one adapter (Read1 acquisition), then sequence with the opposite adapter (Read2 acquisition).

○ Read1 and Read2 from the same DNA fragment can be easily matched since they come from the same cluster.

○ Advantages : Higher accuracy (due to Read1 and Read2 comparison), easy detection of DNA variations, easy analysis of repetitive sequences, and easy mapping between different species.

○ Disadvantages : Higher cost and more steps required than SES.

⑺ WGS (Whole Genome Sequencing)

① SNV, insertion, deletion, structural variant, CNV

② Sequencing depth > 30X

⑻ WES (Whole Exon Sequencing)

① Only SNV, insertion, deletion, SNP in protein-coding genes

② Sequencing depth > 50X ~ 100X

③ Cost-effective

⑼ RNA-seq

① 1^st. Microdissection : Separating specific tissues for RNA extraction.

○ LCM (Laser Capture Microdissection) : Cutting specific tissues with a laser beam. Robust but labor-intensive.

○ TOMO-seq: Using cryosection and computer-based 3D sectioning. Not suitable for clinical purposes.

○ Transcriptome in vivo analysis

○ ProximID

○ STRP-seq

② 2^nd. Attach poly T recognizing the poly A tail of RNA.

③ 3^rd. Fragment RNA.

④ 4^th. Attach primers to RNA.

⑤ 5^th. First cDNA synthesis.

⑥ 6^th. Second cDNA synthesis.

⑦ 7^th. Process the 3’ and 5’ ends of RNA.

⑧ 8th. Ligate DNA sequencing adapters.

⑨ 9th. Amplify ligated fragments with PCR.

⑩ Application 1. dUTP method : A representative method for strand-specific sequencing.

○ Background : Used for studying biological functions based on RNA orientation (e.g., regulation of antisense miRNA).

○ Step 1. DNA &RNA hybrid : Synthesize cDNA (first or anti-sense strand) using dT primers and reverse transcriptase, targeting mRNA poly-A tails.

5’-//-U-//-AAAAAA-3’

3’-//-A-//-TTTTTT-5’

○ Step 2. ds _ cDNA_ : Use dUTP instead of dTTP to synthesize cDNA (first strand) as the template for cDNA (second or sense strand).

3’-//-A-//-TTTTTT-5’

5’-//-U-//-AAAAAA-3’

○ Step 3. ligated _ds cDNA_ : Connect Y-adaptors to both ends of ds cDNA.

○ Step 4. Treatment with UDG (uracil-DNA glycosylase) breaks down the second strand, which contains uracil.

○ Step 5. Amplify the remaining reverse antisense strand (first strand) to create the library.

○ In the library raw data, “_1.fastq” represents the first strand, while “_2.fastq” represents the second strand.

○ Thus, _2.fastq represents the original RNA profile.

⑽ Single-cell sequencing

① Types : scDNA-seq, scRNA-seq (2013 Technology of the Year), single-cell epigenetics sequencing

② Step 1. Isolation of single cells

○ Method 1. Simple isolation : Very early method.

○ Method 2. Based on FACS or LCM (laser microdissection)

○ Method 3. Acoustic separation

○ Separates single cells hydrodynamically, causing minimal impact on cells.

○ CyTOF (cytometry by time of flight) is a representative method.

○ Method 4. Immuno-magnetic separation

○ Attach magnets to cells.

○ Can obtain a large number of cells.

○ Divided into cases with and without centrifugation requirements.

○ Droplet-based platform and plate-based platform have different library size.

③ Step 2. Reverse transcription

④ Step 3. cDNA amplification

⑤ Step 4. Library construction : e.g., Drop-seq

⑥ Single-cell genomics (scDNA-seq) + Single-cell transcriptomics (scRNA-seq)

○ Allows understanding the relationship between genomic mutation patterns and gene expression in transcriptomes.

○ Technologies for separating DNA and RNA : G&T seq, SIDR-seq, DNTR-seq

⑾ Single Nucleus RNA Sequencing (snRNA-seq)

① Purpose 1. Muscles are multinucleated cells, so they need to be analyzed at the nuclear level as they are not captured by scRNA-seq.

② Purpose 2. snRNA-seq captures more various RNA, including introns, pre-mRNA, non-coding RNA, compared to scRNA-seq.

③ Purpose 3. In snRNA-seq, nuclear RNA is primarily captured, while cytoplasmic RNA is also captured (although in small amounts).

⑿ Spatial Sequencing (▶ Supplement)

Figure 8. Overview of spatial sequencing

Table 1. Comparison of different spatial transcriptomic technologies

① Type 1. Spatial genomics

○ Example 1. Tumor research : Tumors are heterogeneous.

○ Example 2. Spleen research : Mature immune cells have diverse genetic compositions.

② Type 2. Spatial transcriptomics : 2020 Technology of the Year

③ 2-1. Spot-based spatial transcriptomics : Many genes + few spots

Figure 9. Betchmark Study of spot-based spatial transcriptomics

○ ST (Spatial Transcriptomics)

○ Barcoded oligos are randomly arranged on a functionalized surface, capturing mRNA released from the mounted tissues and/or cells.

○ 10X Visium

○ Principle : Attach spot-specific oligonucleotides to each spot to hybridize with tissue-derived RNA, obtaining spotwise transcriptomes.

○ Surface area : 6.5 mm × 6.5 mm

○ Thickness : 10 ~ 20 μm

○ Number of spots : Up to 4992 (Based on previous version of Visium HD)

○ Distance between spots : 100 μm

○ Diameter of spots : 55 μm

○ Sensitivity : 10,000 transcripts per spot

○ Type 1. Direct Visium (oligo-dT based method) : Captures mRNAs with poly dT. Only applicable to FF (fresh-frozen) samples.

Figure 10. Principle of Visium FF

○ Type 2. probe-based Visium

○ It can be done in both FF (Fresh Frozen) and FFPE (Formalin-Fixed Paraffin-Embedded) samples. In particular, FFPE (formalin-fixed paraffin-embedded) samples cannot undergo direct Visium due to RNA degradation, where mRNA molecules are fragmented into various pieces.

○ To identify the target mRNA, all three pairs of LHS and RHS must be ligated together: each probe’s length is 25 base pairs. RTL (probe-based RNA-templated ligation chemistry) is utilized for this purpose.

Figure 11. Principle of probe-based Visium

○ Advantage : Superior data quality compared to direct Visium.

○ Disadvantage : Limited freedom in analysis compared to Visium FF, as only genes specified by the probe are detected.

○ For Visium FFPE, starting from June 2024, 10x will discontinue the Visium FFPE service, not using CytAssist.

○ The CytAssist images represent the distribution of gene expression and are used for image alignment.

○ 10X Visium HD

○ The basic data consists of spots with a diameter of 2 μm, and additional data binned at 8 μm and 16 μm are also provided.

○ Slide-seq and Slide-seq V2

○ Employs random spatial bead spreading and in situ sequencing decoding.

○ 97% of spots consist of one or two cell types.

○ HDST

○ Deposits beads with combinatorial barcodes on patterned wafers which are then decoded with serial hybridization.

○ NanoString GeoMx

○ Nanostring lost a patent dispute with 10x Genomics as of Nov ‘23 (ref) → The bankruptsy of Nanostring (ref)

○ Stereo-seq : Higher spatial resolution than Visium

○ Utilizes Illumina or MGI sequencing for oligo patterning on flow cells, and barcode calling is performed directly on the sequencer.

○ Diameter : 220 nm

○ Distance between spots : 500 or 715 nm

○ Seq-Scope : Higher spatial resolution than Visium

○ Utilizes Illumina or MGI sequencing for oligo patterning on flow cells, and barcode calling is performed directly on the sequencer.

○ PIXEL-seq

○ XYZeq

○ Tissue is placed on a spatially barcoded microwell array for an initial round of reverse transcription, after which whole cells are removed and undergo single-cell sequencing.

○ sci-Space

○ Tissue is placed on a glass slide bearing spatially gridded hashing oligos; tissue is then permeabilized to enable oligo transfer and then imaged; nuclei are then extracted, fixed and sequenced.

○ sci-RNA-seq

○ TIVA-seq

○ NICHE-seq

○ ZipSeq

○ It uses patterned illumination and photocaged oligonucleotides to serially print ‘zipcodes’ onto live cells in intact tissues in real time.

○ DBiT-seq

○ Delivers barcoded oligos directly to tissue through orthogonal microfluidics in a predetermined spatial distribution.

○ CITE-seq (ref1, ref2) : Enables parallel comparison of spatial transcriptomics and antibody distribution

Figure 12. Diagram of CITE-seq

○ Connect the 5’ end of oligonucleotide to an antibody using streptavidin-biotin.

○ The oligonucleotide can hybridize complementarily with the oligo-dT primer.

○ Streptavidin-biotin bond can dissociate under reducing conditions.

○ Recently, perturb-CITE-seq was also developed.

○ SPOTS

○ Spatial PrOtein and Transcriptome Sequencing

○ Indirectly assess protein level on Visium using polyadenylated DNA-barcoded antibody

④ 2-2. FISH based spatial transcriptomics : Few genes + many spots

○ ISS( in situ sequencing) : Technique to sequence RNA at its original location in tissue. Sequencing by ligation

○ Type 1. The first ISS

○ Type 2. ISS with Padlock probe

○ Reverse transcriptase creates cDNA of the RNA target

○ Padlock probe can hybridize to two regions of the cDNA

○ Target sequence amplification occurs through RCA (rolling-circle amplification)

○ RCA product is sequenced in situ by ligation

○ Type 3. ISS using fluorescent probes and cross-linking

○ Type 4. barcode based methods

○ Type 5. gap-filled ISS

○ smFISH(single molecule FISH) (2008)

○ seqFISH(sequential FISH) (2014) : DNAse I-based digestion and sequential staining and imaging rounds to decode transcripts

○ seqFISH+ : Genome-scale transcriptome investigation separating individual transcripts into fluorescence spectra, employing 20 probes per encoding round.

○ Vizgen - MERSCOPE (Technology name: MERFISH (multiplexed error-robust FISH))

○ Direct probe hybridization without separate amplification mechanism.

○ Each FISH probe corresponds 1:1 with each gene (though this assumption may not always hold).

○ Employing error correction in barcode assignment for robust barcode calling in noisy FISH-based images.

○ Step 1. Photograph multiple times with fluorescence varying over time for each FISH probe

○ Step 2. Reverse identify genes based on binary code read from each RNA

Figure 13. Principle of MERFISH

○ 10x - Xenium

○ Small amount of padlock probe + rolling circle amplification

○ Step 1. Padlock probe binds complementary RNA transcript in a pincer shape, forming a loop

○ Step 2. RCA (rolling circle amplification) : RNA transcript amplified after loop formation

○ Step 3. Hybridize each RNA transcript with a fluorescent probe, then perform fluorescent imaging → washing

○ Step 4. Repeat Step 3 and decode labels for each gene from the generated images

Figure 14. Principle of Xenium

○ Nanostring - CosMx

○ Small amount of probe + branch chain hybridization

○ Nanostring won in the U.S. against 10x for violating antitrust laws in July ‘23 (ref) → The bankruptsy of Nanostring (ref)

Figure 15. Principle of CosMx

○ FISSEQ and oligoFISSEQ

○ Veranome

○ Rebus

○ BOLORAMIS

○ STARmap : Sequencing by ligation

○ SEDAL sequencing

○ ExSeq

○ BaristaSeq : Sequencing by synthesis

○ BARSeq and BARSeq2

○ HybISS

○ SABER

○ clampFISH

○ split-FISH

○ SCRINSHOT

○ PLISH

○ osmFISH

○ ExFISH

○ par-seqFISH

○ EASI-FISH

○ SGA

○ corrFISH

⑤ Type 3. Spatial proteomics : Broadly classified into mass spectrometry-based and imaging-based methods

○ SWITCH

○ MxIF

○ t-CyCIF

○ IBEX

○ DEI

○ CODEX

○ immuno-SABER

○ TSA

○ Opal IHC

○ MIBI

○ IMC

○ HD-MIBI

○ GeoMx Digital Spatial Profiler (DSP) : 100 mm scale

○ GeoMX DSP stains tissues with suites of antibodies or gene probes fused to UV-cleavable DNA barcodes.

○ 4i multiplexed imaging

⒀ Other sequencing technologies

① TCR-seq (T cell receptor sequencing): Sequencing used to track T cell subtypes and clones.

② Invade-seq: A sequencing technique for analyzing the host-microbiome.

③ long-read sequencing : 2022 Technology of the Year Technology of the Year (Reference)

○ Less sequencing gap compared to short-read sequencing

Figure 16. long-read sequencing and short-read sequencing

○ Advantage 1. AS Analysis(alternative splicing analysis) : Can identify alternative splicing events, isoforms, etc.

○ Advantage 2. Easier integration of epigenetics and transcriptomics

○ Example 1. Pacific Biosciences SMRT (single molecule real-time) sequencing : Average read length is ~20 kb

○ Example 2. Oxford Nanopore Sequencing : Average read length is ~100 kb

④ non-invasive sequencing

○ A technology that allows sequencing without breaking cells

⑤ Halo-seq : A technique for obtaining the transcriptome of RNAs adjacent to a specific protein.

○ Step 1. Attach a HaloTag domain to a specific target.

○ Step 2. This HaloTag generates an alkyne handle radical by ejecting a hydrogen radical H· from a radical-producing Halo ligand injected with an alkyne handle.

○ R-H → R· + H·

○ Step 3. Similarly, the HaloTag generates an RNA radical by ejecting a hydrogen radical H· from RNA.

○ RNA-H → RNA· + H·

○ Step 4. The alkyne handle radical combines with the RNA radical.

○ Step 5. React alkyne-RNA with biotin azide to produce biotinylated RNA.

○ Step 6. Separate only the biotinylated RNA using affinity chromatography with streptavidin.

○ Step 7. RNA-seq allows for the detection of RNAs close to the specific target.

○ Reason : Radicals are unstable and cannot travel long distances.

Figure 17. Principle of Halo-seq

⑥ multi-NTT seq (nanobody tethered transposition followed by sequencing)

⑦ Epigenomics Sequencing(epigenomics sequencing)

⑧ 3D Sequencing

⑨ Temporal Sequencing

○ Record-seq

○ Live-seq

○ TMI

○ molecular recording

⑩ Spatiotemporal Omics

○ ORBIT (single-molecule DNA origami rotation measurement)

○ 4D spatiotemporal MRI or hyperpolarized MR

○ in vivo 4D omics with transparent mice

⒁ NGS (next-generation sequencing) Summary

① Cost of genome analysis

○ 2001 : Human Genome Project benchmark $100 million / person

○ 2007 : 100 billion won / 4 years

○ 2008 : 454 Life Sciences standard $1,000,000 / person. 1.5 billion won / 4.5 months

○ 2009 : Helicos BioSciences standard $48,000 / person

○ Predicted to be sufficient with one million won by 2014 (Nature 456, 23-25, 2008)

② Scale of genome analysis

Figure 18. Trend of genome analysis scale

③ Relationship between depth and coverage

○ sequencing depth (read depth) : Indicates how many times a specific nucleotide appears on average

Figure 19. Definition of depth

○ “10x” means it was read 10 times

○ Can be defined for each nucleotide

○ coverage (c)

○ c : = LN / G

○ L : read length

○ N : number of reads

○ G : haploid genome length

○ Comparison of depth and coverage

○ Sequencing depth represents total read number

○ Coverage represents the relationship between sequence reads and reference (e.g. whole genome, al locus)

○ Otherwise, depth and coverage are very similar concepts

④ Relationship between bulk and read

○ bulk : total RNA production

○ In case of equal depth, as bulk increases, RNA read count is inversely proportional, causing irrationality

○ Example: In spatial transcriptomics, bulk is typically large and depth is low, resulting in low RNA read count

○ Normalization: Various methods have been introduced to resolve this irrationality

⑤ Relationship between read count and number of reads

○ If read length is less than 250 bp, it is impossible to detect sequence error

○ Relationship between read length and number of reads per run: there is a trade-off

Figure 20. Relationship between read length and number of reads per run

⑥ Relationship between transcriptome read count and gene expression

○ read count : Actual number of transcripts

○ gene expression : Value corrected from read count through normalization process

Entered : 2015.07.02 23:31

Updated : 2022.03.13 13:11

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Chapter 10. Genome Project and Sequencing Technology

1. Genome Project

2. Sequencing Technology

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