cytogenomics tools and techniques

1. Submitted by :-
P.TEJASREE
TAD/2023-010
Ph.D 1st Year
Dept. of GPBR
Cytogenomics tools and techniques,
Chromosome sorting
Submitted to :-
Dr. M. Sreevalli Devi
Assistant Professor
Dept. of GPBR 1
ACHARYA N.G. RANGA AGRICULTURAL
UNIVERSITY
S.V. AGRICULTURAL COLLEGE, TIRUPATI

2. Cytogenomics:
Whole
genome
sequencing
Structural
variations
Palaeopolyploidy
Synthesis of
microbial genomes
and Eukaryotic
chromosomes
3D
Chromosome
conformations
(chromosomics)

3. Molecular karyotyping
Both karyotyping and microarray-based comparative genomic
hybridization are methods to identify gains and losses throughout the
genome in order to provide an explanation for an abnormal phenotype.
Molecular karyotyping, also “array-comparative genomic
hybridization” (aCGH) or “chromosomal microarray” (CMA)

4. Molecular combing
DNA-replication, replication kinetics, but also for copy number variations of satellite sequences
down to single nucleotide polymorphisms (SNPs) are possible and can be visualized
MCT uses the force exerted in the process of liquid flow via surface tension to stretch DNA molecules and
spread them on solid surfaces, i.e. glass cover slips; most stretched DNA-fibers; FISH probes can be
hybridized and basic studies on DNA-replication

5. Chromosome Orientation-FISH (CO-FISH)
CO-FISH has been successfully applied to study orientation of repeated sequences or long unique DNA sequences
The procedure works by culturing cells for a single round of replication in the presence of the thymidine analog 5-bromo-2′-deoxyuridine
(BrdU), thus incorporating BrdU into the newly synthesized daughter strands. The daughter strands are then specifically removed through
nuclease digestion, leaving single-stranded chromosomal DNA, which is an ideal target for hybridization of single-stranded probes.
A sister chromatid exchange within telomeric DNA (T-SCE) will lead
to combined green and red fluorescence.

6. Telomeres Accessed by Q-FISH
In Q-FISH, the technique uses labelled
(Cy3 or FITC) synthetic DNA mimics called peptide
nucleic acid (PNA) oligonucleotides to quantify
target sequences in chromosomal DNA
using fluorescent microscopy and analysis software.
Q-FISH is most commonly used to
study telomere length

7. Parental Origin Determination
FISH (POD-FISH)
Taking advantage of CNVs, the same can be done in trio-analyses of
chromosome preparations. parental origin determination that relies on
the design of CNV-based sets of probes for fluorescence in situ
hybridization (POD-FISH). Apart from uniparental disomy testing, by
this approach (called parental origin determination FISH - PODFISH)
also the inheritance of individual chromosomes can be visualized.
Distinction between maternal (normal- left) and paternal
(translocated-right) child’s chromosomes 1 has been possible
because the secondary constriction in the father’s chromosome
was always shorter than in that of the mother

8. Multi color FISH (m-FISH)
m-FISH implemented primarily to study acquired or inherited chromosomal aberrations in diagnostics
Multicolor FISH (M-FISH) assays are used for a precise assessment of complex chromosomal rearrangements. This
technique uses all whole-chromosome painting probes. Thus, complex chromosomal rearrangements, and all
numerical aberrations can be visualized simultaneously in a single hybridization experiment

9. Spectral karyotyping
Spectral Karyotyping (SKY) is a cytogenetics technique used in studying genomic and
chromosomal compositions. This technique takes advantage of chromosome painting
probes, and the detection of these probes are acquired through a sagnac interferometer.
Autosomal Dominant Polycystic Kidney Disease

10. centromere-specific Multicolor-FISH (cenM-FISH)
supernumerary marker chromosomes
(SMC) found in prenatal and postnatal
diagnosis are usually small abnormal
chromosomes that appear in addition to
the 46 normal chromosomes. They are
also called extra structurally abnormal
chromosomes, accessory or B-
chromosomes.
useful technique for the characterization
of complex chromosomal aberrations
labeled centromeric satellite DNA as
probes. This approach allows the rapid
identification of all human centromeres by
their individual pseudo-coloring in one
single step

11. Chromoanagenesis - chromothripsis, chromoanasynthesis and chromoplexy
• Chromothripsis refers to the localized
shattering and reshuffling of one or a
few chromosome segments during a
one-step catastrophic event, with the
incomplete repair of double-strand
breaks (DSBs) through non-
homologous end-joining (NHEJ).
• Chromoplexy involves a series of
chained, complex inter- and intra-
chromosome translocations
including up to eight
chromosomes and presumably
occurring simultaneously
• Chromoanasynthesis arise from the
defective replication of a single or a
few chromosomes mediated by fork-
stalling and template switching
(FoSTeS) or microhomology-
mediated break-induced replication
(MMBIR) processes.

12. Chromosomal Heteromorphisms and Repetitive DNA-Elements
These CHMs, consisting mainly of repetitive DNA-elements,
like satellite DNAs, are definitely understudied.
probes like DXZ1 and DYZ3 are commercially available
centromere specific probes for chromosomes X and Y

13. glass-needle
based
chromosome
microdissecti
on (midi) - 1

14. glass-needle based
chromosome microdissection
(midi) - 2

15. glass-needle based chromosome microdissection (midi) - 3
picogram of DNA can be taken directly from
chromosomes and studied in multiple ways afterwards,
including NGS approaches and others. Also prior FISH-
labelled metaphases can be applied in midi, which can
help to extract the correct (part of a) chromosome
cut out a chromosomal band/region or scrape together one
whole chromosome to a single spot. Afterward pick up the
microdissected DNA by the glass needle. Then the DNA
sticking to the glass needle is transferred to the collection drop
in the pipette and washed off.
The DNA accumulated in the collection drop after 1–15 midi
steps is then transferred into a reaction tube. This is subjected
to DNA probe amplification by a specific PCR procedure
[degenerate oligonucleotide primed- polymerase chain reaction
(DOP-PCR)]
most cost-efficient way to establish whole or partial
chromosome painting probes for molecular cytogenetic
studies or other studies including sequencing

16. Comet FISH assay -1

17. Comet FISH assay -2
measuring DNA damage, protection, and repair at the level of individual
cells. In this assay, cells are embedded in agarose, lysed, and then
electrophoresed. Negatively charged broken DNA strands exit from the
lysed cell under the electric field and form a comet with “head” and
“tail”. The amount of DNA in the tail, relative to the head, is proportional
to the amount of strand breaks. Results from the comet assay alone reflect
only the level of overall DNA damage in single cells. FISH permitted to
supplement potential of the comet assay with an opportunity to recognize
genome regions of interest on comet images

18. CRISPR-FISH
RNA-guided endonuclease-in situ labeling (RGEN-ISL)

19. Chromosome Conformation Capture Techniques
three-dimensional genome organization can be studied on single cell level either by single-cell Hi-C or by imaging based approaches

24. a) Populations with disrupted epigenomes can be created through crossing DNA methylation mutants to wild-type parents and segregating away the mutant allele.
These have been used to create biparental epigenome-recombinant inbred line (epiRIL) populations in Arabidopsis to identify cryptic alleles and mapping of
epigenome-quantitative trait locus (epi-QTL). B) DNA methylation profiling in natural (or induced) populations can be used to identify epigenomic variants
(epialleles). These can be used along with phenotypic variation to carry out epigenome-wide association studies (Epi-WAS) to identify candidate loci or epi-markers.
C) Stress-induced epigenetic changes could be inherited meiotically and passed onto future generations or mitotically, which can prime plants for future stress
exposure. These can be used to create plants with increased stress tolerance.
EPIGENETICS
The stability and
memory features of
epigenetic variations
have to be carefully
considered for
breeding applications.

25. DNA methylation. The process
involves attaching a methyl group to
the 5th carbon of cytosine in DNA. In
plants, methylation occurs in three
sequence contexts: CG, CHG, and
CHH, where H = A/T/G.
Modifications of histones, involve post-
translational modifications of the histone
tails, mainly methylation and acetylation.
It is assumed that on histone H3,
methylation of lysine 4 and 36 and
acetylation of lysine 4 and 9 is responsible
for enhancing gene expression, while
methylation of lysine 9 and 27 contributes
to the inhibition of the expression of
selected genes
Non-coding RNAs. Small, about 20
nucleotide RNA molecules that do not
code proteins, but regulate gene
expression. They can participate in the
RNA-directed DNA methylation (RdDM)
pathway—small interfering RNA
(siRNA), or by inducing a silencing
complex (RISC) and binding to
complementary genes on DNA, causing
their silencing (miRNA).
Epigenetic changes contributing to the plasticity characteristics in a plant to stressful environmental conditions

26. Sequencing technologies

27. Genome projects timeline

28. Cumulative number of eukaryotic families for which assemblies were generated by EBP affiliates over time
Earth biogenome project

31. (a) Depicts a whole genome shotgun procedure starting by fragmenting the genome into pieces (≥10 kb), sequencing the
fragments and ending with the assembly based on sequence overlaps.
(b) (b) Shows the hierachical approach utilizing a large insert BAC library (up to 200 kb), a minimal set of overlapping BACs
and the subsequent shotgun sequencing of the selected BACs

32. Optical mapping
Optical mapping is a technique for constructing ordered,
genome-wide, high-resolution restriction maps from single,
stained molecules of DNA, called "optical maps“;
highlighting copy number and structural anomalies, including
balanced translocations.

33. Development of pan genomes
A pangenome aims to capture the complete genetic diversity within a
species and reduce bias in genetic analysis inherent in using a single
reference genome.

35. Personal genomics
Personal genomics is an area of genomics focused on an
individual's genome sequencing - this personal genome
diagnostic helps to analyze a person's biology and provide
information for potential health plans and treatments.

36. Comparative genomics
Comparative genomics can be used to define important structural sequences that are
identical in many genomes and to detect evolutionary changes across genomes.

37. Orthology and paralogy
Orthologs, or orthologous genes, are genes in different species that originated
by vertical descent from a single gene of the last common ancestor
Genes separated by speciation are called orthologs.
Genes separated by gene duplication events are called paralogs.
Homologous sequences are paralogous if they were separated by a gene
duplication event: if a gene in an organism is duplicated to occupy two
different positions in the same genome, then the two copies are paralogous.

38. Structural variations

40. Read depth . Reads are aligned into the reference genome and when compared to diploid regions they show a reduced number of reads in a
deleted region or higher read depth in a duplicated region.
Paired reads. Pairs of sequence reads are mapped into the reference genome (from left to right): (1) no SV, pairs are aligned into correct order,
correct orientation and spanned as expected based on the library’s insert size; (2) deletion, the aligned pairs span far apart from that expected based
on library insert size; (3) tandem duplication, read pairs are aligned in unexpected order, where expected order means that the leftmost read should
be aligned in the forward strand and the rightmost read in the reverse strand; (4) novel sequence insertion, the pairs are aligned closer from that
expected based on library insert size; (5) inversion, read pairs are aligned in wrong orientation, both reads align either in forward or reverse strand;
and (6) read pairs mapped to different chromosomes.

41. Split reads. Sequenced reads pointing to the same
breakpoint are split at the nucleotide where the breakpoint
occurs. The corresponding paired read is properly aligned
to the reference genome.
De novo assembly. Sample reads from novel
sequence insertions are assembled without a
reference sequenced genome.

42. Ka/Ks ratio

43. Oligo array for CGH
CGH uses two genomes, a test and a control, which are
differentially labeled and competitively hybridized
to metaphase chromosomes. The fluorescent signal
intensity of the labeled test DNA relative to that of the
reference DNA can then be linearly plotted across each
chromosome, allowing the identification of copy number
changes

44. SNP array and hybridization intensities
First, hybridization is performed on a single sample per
microarray, and log-transformed ratios are generated by
clustering the intensities measured at each probe across many
samples
key advantage of SNP microarrays is the use of SNP allele-
specific probes to increase CNV sensitivity, distinguish alleles
and identify regions of uniparental disomy through the
calculation of a metric termed B allele frequency

45. Paleopolyploidy, which refers to ancient whole-genome duplication events in the evolutionary history of a species
Paleopolyploidy
Such an event could either double the genome of a single species (autopolyploidy) or combine
those of two species (allopolyploidy).
Because of functional redundancy, genes are rapidly silenced or lost from the duplicated genomes. Most paleopolyploids,
through evolutionary time, have lost their polyploid status through a process called diploidization, and are currently
considered diploids

46. Tree based Synteny blocks

47. Synthesizing genomes
PCAASSEMBLY
Polymerase cycling
assembly (or PCA, also known
as Assembly PCR) is a method for
the assembly of
large DNA oligonucleotides from
shorter fragments. The process uses
the same technology as PCR, but
takes advantage of DNA
hybridization and annealing as well
as DNA polymerase to amplify a
complete sequence of DNA in a
precise order based on the single
stranded oligonucleotides used in the
process. It thus allows for the
production of synthetic genes and
even entire synthetic genomes.

48. Gibson assembly, described by Gibson et al. allows for single-step isothermal assembly of DNA with up to several
hundreds kb. By using T5 exonuclease to 'chew back' complementary ends, an overlap of about 40bp can be created. The
reaction takes place at 50 °C, a temperature where the T5 exonuclease is unstable. After a short timestep it is degraded, the
overlaps can anneal and be ligated

49. Synthetic Chromosome Rearrangeme
nt And Modification By LoxP-
mediated Evolution or SCRAMBLE.
Through SCRaMbLE, massive rearrangements
can be introduced into the synthetic
chromosomes, including intra-chromosomal
deletions, inversions, duplications, and inter-
chromosomal translocations
It is these loxP sites, which facilitate the
recombination of genetic elements in either
direction by the Cre recombinase, that allow
the synthetic chromosomes to be used for
applications that require large amounts of
genetic diversity.

50. Chromosome sorting: Isolation of
specific chromosome for
development of molecular maps and
gene location

51. Overview of the three technologies used in this study
(flow cytometry capture, laser capture
microdissection, magnetic streptavidin-bead capture).

52. (A)For flow cytometry capture, the extracted chromosomes were incubated with a specific biotin Y chromosome
probe and stained with streptavidin-PE and DAPI. Y chromosomes were then sorted in a FACSAria flow
cytometer. The sorted chromosomes were collected in Eppendorf (Hamburg, Germany) tubes in ddH2O
(double-distilled water) for further processing.
(B)For laser capture microdissection, individual chromosomes were hybridized with Y chromosome-specific
probes conjugated with FITC (green), counterstained with DAPI (blue) and mounted on slides covered by
polyethylene membranes. On these slides, they were selected and catapulted by the laser pressure catapulting
(LPC) function in a Zeiss PALM MicroBeam IV Laser Microdissector. Y chromosomes were captured within
the cap and dissolved in TE buffer. The cap was closed and the sample was spun down by centrifugation.
(C)For magnetic streptavidin-bead capture, chromosomes were incubated with a specific biotin Y chromosome
probe as in the previous procedure. Dynabeads MyOne streptavidin beads were added to the probe Y
chromosome mixture and magnetic separation was performed to capture the Y chromosome on a magnetic
rack. Finally, in all cases, physical fragmentation was performed before library prep and sequencing with a
Covaris S2 sonicator.

53. • The separation of any chromosome complement depends very much upon the DNA stain used and the laser
power available to excite the stain.
• Ethidium bromide stains all DNA base pairs nonspecifically, but Hoechst 33258 preferentially stains A-T
basepairs and chromomycin A3 stains G-C basepairs.
• Large lasers are essential for separating chromosomes on the basis of the complementary DNA stains Hoechst
and chromomycin A3.
• Sorting at fast flow rates (2200/sec) with 18-watt argon lasers requires considerably less sorting time than
sorting at slower flow rates (150/sec) with 5-watt argon lasers for 16 hrs provides chromosomal DNA enriched
from 12 pg of total DNA which is enough to determine reliably the unique gene content of the two sorted
fractions by restriction enzyme analysis.
• The DNA fragments are separated according to size by agarose gel electrophoresis and transferred to
nitrocellulose filter paper. The DNA sequences of interest are identified following hybridization of the filter to
radiolabeled DNA probe by autoradiography.

54. Gene mapping
• Adjacent DNA sequences may be characterized by studying overlapping cloned DNA sequences
• DNA libraries constructed from sorted chromosomes may be used to locate additional linked unique DNA sequences
• The entire chromosome complement can be screened in two sorts. The large gene-containing chromosome group is further
fractionated to identify the single peak that contains the gene of interest. Finally, different sized abnormal chromosomes that
contain segments of chromosomes in the peak of interest are sorted from the normal homologous chromosome.
• Current chromosome sorting techniques could be used to separate rearranged and normal chromosomes in several species.
• Libraries of enriched sorted chromosome fractions may be constructed on nitrocellulose filter paper no matter what the
sorting resolution. These sorted DNA fractions may then be hybridized up to 5 times with more than one unique probe each
time to determine the gene content of the chromosome fractions.
• These fractions are then subfractionated several more times to locate the gene within the smallest possible fraction of normal
chromosomes. Rearranged chromosomes are then sorted to test for the gene in a new fraction.
Gene mapping by in situ hybridization
Gene mapping by in situ hybridization involves direct hybridization of radiolabeled probes to metaphase cells fixed on
microscope slides. Hybridized radioactive probe is detected by a photographic emulsion applied directly to the slide.

55. Insulin gene mapping

56. References
• Thomas Liehr.2021.Molecular Cytogenetics in the Era of
Chromosomics and Cytogenomic Approaches
• Cytogenetics – P.K Gupta
• Cytogenomics - Thomas Liehr
• Roger V. Lebo.1982. Chromosome Sorting and DNA Sequence
Localization