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Comparison of mouse and man at genome level
Material of this lecture taken from the paper of the Mouse Genome Sequencing
Consortium „Initial sequencing and comparative analysis of the mouse genome“,
Nature 420, 520-562 (5.12.2002). Excellent paper! Well readable!
Key findings:
* the mouse genome is about 14% smaller than the human genome. The
difference probably reflects a higher rate of deletion in mouse.
* over 90% of the mouse and human genomes can be partitioned into
corresponding regions of conserved synteny (segments in which the gene order
in the most recent common ancestor has been conserved in both species)
* at the nucleotide level, ca. 40% of the human genome can be aligned to the
mouse genome. These sequences seem to represent most of the orthologous
sequences that remain in both lineages from the common ancestor. The rest
was probably deleted in one or both genomes.
* the neutral substitution rate has been roughly half a nucleotide substitution per
site since the divergence of the species. About twice as many of these
substitutions have occurred in mouse as in human.
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Comparison of mouse and man at genome level
Key findings:
* the proportion of small (50-100 bp) segments in the mammalian genome that is
under (purifying) selection is ca. 5%, i.e. much higher than can be explained by
protein-coding sequences alone.
 genome contains many additional features (UTRs, regulatory elements, nonprotein-coding genes, chromosomal structural elements) under selection for
biological function!
* the mammalian genome is evolving in a non-uniform manner, various measures
of divergence showing substantial variation across the genome.
* mouse and human genomes each seem to contain ca. 30.000 protein-coding
genes. The proportion of mouse genes with a single identifiable orthologue in the
human genome is ca. 80%. The proportion of mouse genes without any homologue
currently detectable in the human genome (and vice versa) is < 1%.
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Key findings 3
* dozens of local gene family expansions have occurred in the mouse lineage:
genes related to reproduction, host defence, immune response
* despite marked differences in activity of transposable elements, similar types of
repeat sequences have accumulated in the corresponding genomic regions.
* by additional sequencing in other mouse strains, about 80.000 SNPs identified.
Distribution of SNPs shows that genetic variation among mouse strains occurs in
large blocks.
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Background of mouse sequencing project
Origins of the mouse
human and mouse lineages probably diverted 65 million years ago
Origins of mouse genetics
already ancient Chinese references about mice with different colors
by the 1700s mouse fanciers in Japan and China had domesticated many varieties
as pets :-) Europeans subsequently imported favourites and bred them to local
mice.
Genetic mapping in the mouse began in 1915.
Origins of mouse genomics
mouse was included as of five central model organisms in Human Genome Project.
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Generating the draft genome sequence
Consortium selected strain B6
Sequencing strategy
- first whole genome shotgun to generate draft sequence quickly
- later generate physical map for producing a finished sequence
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Sequencing
- paired-end reads of different length!
- 33.6 Million of 41.4 Million reads of sufficient quality
- ca. 7-fold coverage
The mouse genome. Nature 420, 520 - 562
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Anchoring + Assembly
- 2 assembly programs used: Arachne and Phusion. Comparable outputs.
- most of the genome lies in supercontigs that are extremely large;
the 200 largest supercontigs span more than 98% of the assembled sequence.
- when compared to mouse genetic map no evidence for incorrect global joins
within the supercontigs
The mouse genome. Nature 420, 520 - 562
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Chromosomal structure
The mouse genome in 88 sequence-based
ultracontigs. The position and extent of the 88
ultracontigs of the MGSCv3 assembly are shown
adjacent to ideograms of the mouse
chromosomes. All mouse chromosomes are
acrocentric, with the centromeric end at the top of
each chromosome. The supercontigs of the
sequence assembly were anchored to the mouse
chromosomes using the MIT genetic map.
Neighbouring supercontigs were linked together
into ultracontigs using information from single BAC
links and the fingerprint and radiation-hybrid maps,
resulting in 88 ultracontigs containing 95% of the
bases in the euchromatic genome.
The mouse genome. Nature 420, 520 - 562
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Genome size
total length 2.5 Gb
The mouse genome. Nature 420, 520 - 562
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Quality assessment
at intermediate scale:
- compare positions of well-studied markers on the mouse genetic map and
in the genome assembly. 2605 markers were unambiguously mapped
(E-value in BLAST of < 10-100 to a single location).
Conflicts in 1.8% of the cases; typical accuracy of genetic maps.
11 cases investigated. In 10 cases, remapping of genetic map resolved problem.
1 case left: a 36 kb segment that was merged into the wrong contig.
at fine scale:
align genome to 10 Mb of finished BAC-derived sequence from B6 strain.
39 discrepancies of > 50 bp in length (median size of 320 bp) reflecting
small misassemblies.
Discrepancies typically occur at the ends of contigs in WGS assembly 
incorrect incorporation of a single terminal read.
The mouse genome. Nature 420, 520 - 562
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Unplaced reads and large tandem reads
One expects that highly repetitive regions of the genome will not be assembled
or anchored on the chromosomes.
Indeed, 5.9 million of the 33.6 million reads were not part of anchored sequence.
88% of them are not assembled into sequence contigs, 12% belong to contigs
but are not localized on a particular chromosome.
A striking example: large region on Chr1 that contains a tandem expansion of
sequence containing a Sp100-rs gene fusion.
Region is highly variable, even among laboratory strains – estimated lengths
ranging from 6 – 200 Mb.
Bulk of this region not reliably assembled. From the sequence reads one
estimates 493-fold coverage of Sp100-rs gene suggesting that there are ca.
60 copies in the B6 genome.
(Consistent with estimate of 50 copies obtained by Southern blotting.)
The mouse genome. Nature 420, 520 - 562
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End of Assembly section –
Now starts Analysis!
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Conservation of synteny between human and mouse
Starting from a common ancestral genome approximately 75 Million years
ago, human and mouse genomes have each been shuffled by chromosomal
rearrangements. The rate of these changes is low enough that local gene
order remains largely intact.
In their pioneering paper, Nadeau and Taylor, 1984 estimated that the mouse
and human genomes could be parsed into roughly 180 syntenic regions – a
surprisingly small number.
Today, gene-based syteny maps define about 200 syntenic regions.
The mouse genome. Nature 420, 520 - 562
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Detect syntenic regions with PatternHunter
- perform sequence comparison of entire mouse and human genome sequences
to identify regions with a high similarity score > 40 (corresponding to a 40-base
perfect match with penalties for mismatches and gaps)
- also require that each sequence is the other‘s unique match above this
threshold.
Such regions probably reflect orthologous sequence pairs.
About 558.000 pairs found! Mean spacing of 4.4 kb; N50 length of ca. 500 kb.
Together they make up 7.5% of the mouse genome.
But there may be many more that have evolved too quickly to be detected.
The mouse genome. Nature 420, 520 - 562
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Identify regions of conserved synteny
Syntenic segment: maximal region in which a series of landmarks occur in the
same order on a single chromosome in both species.
Syntenic block: one or more syntenic segments that are all adjacent on the same
chromosome in human and on the same chromosome in mouse; may otherwise
be shuffled with respect to order and orientation.
(only consider regions > 300 kb)
Each genome could be parsed into a total of 342 conserved syntenic segments.
On average, each landmark resides in a segment containing 1600 other
landmarks.
Segments vary greatly in length: 303 kb – 64.9 Mb.
About 90.2 % of human and 93.3% of mouse genome unambigously reside
with conserved syntenic segments.
The mouse genome. Nature 420, 520 - 562
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Conservation of synteny between human and mouse
A typical 510-kb segment of mouse chromosome 12 that shares common
ancestry with a 600-kb section of human chromosome 14 is shown. Blue lines
connect the reciprocal unique matches in the two genomes. The cyan bars
represent sequence coverage in each of the two genomes for the regions. In
general, the landmarks in the mouse genome are more closely spaced,
reflecting the 14% smaller overall genome size.
The mouse genome. Nature 420, 520 - 562
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Correspondence of syntenic regions
Segments and blocks >300 kb in size with conserved synteny in human are
superimposed on the mouse genome. Each colour corresponds to a particular
human chromosome. The 342 segments are separated from each other by thin,
white lines within the 217 blocks of consistent colour.
The mouse genome. Nature 420, 520 - 562
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Dot plots of conserved syntenic segments
For each of three human (a–c) and mouse (d–f)
chromosomes, the positions of orthologous
landmarks are plotted along the x axis and the
corresponding position of the landmark on
chromosomes in the other genome is plotted on
the y axis. Different chromosomes in the
corresponding genome are differentiated with
distinct colours. In a remarkable example of
conserved synteny, human chromosome 20 (a)
consists of just three segments from mouse
chromosome 2 (d), with only one small segment
altered in order. Human chromosome 17 (b) also
shares segments with only one mouse
chromosome (11) (e), but the 16 segments are
extensively rearranged. However, most of the
mouse and human chromosomes consist of
multiple segments from multiple chromosomes, as
shown for human chromosome 2 (c) and mouse
chromosome 12 (f). Circled areas and arrows
denote matching segments in mouse and human.
The mouse genome. Nature 420, 520 - 562
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Sytenic properties of human and mouse chromosomes
The mouse genome. Nature 420, 520 - 562
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Size distribution of elements with conserved synteny
Size distribution of segments and blocks with synteny conserved between
mouse and human. a, b, The number of segments (a) and blocks (b) with
synteny conserved between mouse and human in 5-Mb bins (starting with
0.3–5 Mb) is plotted on a logarithmic scale. The dots indicate the expected
values for the exponential curve of random breakage given the number of
blocks and segments, respectively.
The mouse genome. Nature 420, 520 - 562
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Genome rearrangement?
Using the methods from lecture 5 (Pevzner & Tesler algorithms) one can
compute the minimal number of rearrangements needed to „transform“ one
genome into the other.
When applied to the 342 syntenic segments, the most parsimonious
(=shortest) path has 295 rearrangements.
The analysis suggests that chromosomal breaks may have a tendency to
reoccur in certain regions.
With only two species, however, it is not yet possible to recover the ancestral
chromosomal order or reconstruct the precise pathway of rearrangements.
This will become possible in short time as more and more mammalian species
are sequenced.
The mouse genome. Nature 420, 520 - 562
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Next: Genome landscape
- genome expansion and contraction
What accounts for the smaller size of the mouse genome? See section on
repeats.
- (G + C) content
In mammalian genomes, there is a positive correlation between gene density
and (G + C) content.
- CpG islands
The mouse genome. Nature 420, 520 - 562
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(G + C) content
The overall distribution of local (G + C)
content is significantly different between
the mouse (blue) and human (red) genomes.
In human, 1.4% of the windows have
(G + C) > 56% and 1.3% with < 33%.
Such extreme deviations are absent in the
mouse genome.
The reason for this difference is unknown.
Both species have 75-80% of genes residing in the (G+C)-richest half of
their genome (see below).
Mouse shows similar extremes of gene density despite being less extreme
in (G+C) content.
The mouse genome. Nature 420, 520 - 562
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CpG islands
In mammalian genomes, the palindromic dinucleotide CpG is usually methylated
on the cytosine residue. Methyl-CpG is mutated by deamination to TpG,
leading to ca. 5-fold underrepresentation of CpG across the human and mouse
genomes.
In some genome regions that have been implicated in gene regulation, CpG
dinucleotides are not methylated.
Such regions, termed CpG islands are usually a few hundred Nt in length, have
high (G + C) content and above average representation of CpG dinucleotides.
Search genomes with program: detect regions on basis of (G + C) and CpG
content.
 Mouse genome contains fewer CpG islands (15.500) than human (27.000).
The mouse genome. Nature 420, 520 - 562
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CpG islands
(G+C) content and density of CpG islands shows more variability in human (red)
than mouse (blue) chromosomes. a, The (G+C) content for each of the mouse
chromosomes is relatively similar, whereas human chromosomes show more
variation; chromosomes 16, 17, 19 and 22 have higher (G+C) content, and
chromosome 13 lower (G+C) content. b, Similarly, the density of CpG islands is
relatively homogenous for all mouse chromosomes and more variable in human,
with the same exceptions. Note that the mouse and human chromosomes are
matched by chromosome number, not by regions of conserved synteny.
The mouse genome. Nature 420, 520 - 562
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Repeats
Repeats are the most prevalent feature of mammalian genomes.
Most of them are interspersed repeats representing „fossils“ of transposable
elements.
Transposable elements are a principal force in reshaping the genome.
Their fossils provide powerful reporters for measuring evolutionary forces
on the genome.
About 46% of the human genome can be recognized currently as interspersed
repeats resulting from transposable elements during the past 150 – 200
million years.
The total fraction derived from transposons could be considerably larger
but fossils older than a certain age cannot be detected anymore due to the
high degree of sequence divergence.
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Repeats
All mammals have essentially the same 4 classes of transposable elements:
1 LINE: autonomous long interspersed nucleotide element
2 SINE: LINE-dependent, short RNA-derived short interspersed nucleotide elements
3 LTR: retrovirus-like elements with long termain repeats
1 - 3 procreate by reverse transcription of an RNA intermediate
4 DNA transposons; move by a cut-and-paste mechanism of DNA sequence
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Interspersed repeats
32.4% (mouse) of genome are lineage-specific repeats vs. 24.4% for human
The mouse genome. Nature 420, 520 - 562
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Interspersed repeats
Mouse lacks ancestral repeats; they comprise only 5% of the mouse genome
vs. 22% of the human genome.
Median divergence levels of 18 subfamilies of interspersed repeats that were
active shortly before the human-rodent specification indicates an approximately
twofold higher average substitution rate in mouse than in human.
Comparison of ancestral repeats to their consensus sequence also allows an
estimate of the rate of occurrence of small (<50 bp) insertions and deletions.
Both species show a net loss of nucleotides. The overall loss due to small
indels in ancestral repeats is at least twofold higher in mouse than in human.
(This contributes ca. 1-2% to the smaller size of the mouse genome).
This is an average. Currently, the substitution rate per year in mouse is probably
fivefold higher than in human.
The mouse genome. Nature 420, 520 - 562
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Density of interspersed repeat classes
In both species, there is a strong
increase in SINE density and a
decrease in L1 density with
increasing (G+C) content, with
the latter particularly marked in
the mouse. Another notable
contrast is that in mouse, overall
interspersed repeat density
gradually decreases 2.5-fold with
increasing (G+C) content,
whereas in human the overall
repeat density remains quite
uniform.
The mouse genome. Nature 420, 520 - 562
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Similar repeats accumulate in orthologous locations
Contrast in the genomic distribution of LINEs and SINEs:
Whereas LINES are strongly biased towards (A + T) regions, SINEs are
strongly biased towards (G + C) rich regions.
Are (A + T) and (G + C) truly causative factors or merely reflections of an
underlying biological process?
Interpreation of analysis: SINE density is influenced by genomic factors that are
correlated with (G + C)-content but that are distinct from (G + C) content per se.
The mouse genome. Nature 420, 520 - 562
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Mouse genes
Evidence-based gene prediction
1
ENSEMBL gene prediction pipeline:
- know protein-coding cDNAs are mapped onto the genome
- additional protein-coding genes are predicted on the basis of
similarity to proteins in any organism using GeneWise.
- consider all those de novo gene predictions from GENSCAN
that are supported by experimental evidence (such as ESTs)
2
augment with Genie gene prediction pipeline
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Predicted Mouse genes
The mouse genome. Nature 420, 520 - 562
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Pseudogenes
An important thing in annotating mammalian genomes is distinguishing real
genes from pseudogenes (inactive gene copies).
Processed pseudogenes arise through retrotransposition of spliced or partially
spliced mRNA into the genome; they are often recognized by the loss of some
or all introns relative to other copies of the gene.
Unprocessed pseudogenes arise e.g. from duplication of genomic regions.
They sometimes contain all exons, but often have suffered deletions and
rearrangements.
Over time, pseudogenes of either class tend to accumulate mutations that clearly
reveal them to be inactive, such as multiple frameshifts or stop codons.
They acquire a larger ratio of non-synonymous to synonymous substitutions
(KA / KS) than functional genes.
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Gapdh gene: example of a pseudogene
The mouse genome contains only a single functional Gapdh gene,
but there are > 400 pseudogenes distributed across 19 of the mouse
chromosomes.
Some these are readily identified as pseudogenes, but 118 have retained
enough genic structure that the could be identified as pseudogenes only
by manual inspection!
Suspicious genes are:
(1) genes that lack a corresponding gene prediction in the region of conserved
synteny in the human genome (2705)
(2) genes that are members of apparent local gene clusters and that lack a
reciprocal best match in the human genome (5143).
Authors estimate that 76% of first class and 30% of second class are pseudogenes. They comprise ca. 12000 exons in the 213562 mouse gene catalogue.
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Comparison of mouse and human gene sets
Approximately 99% of mouse genes have a homologue in the human genome.
For 96% the homologue lies within a similar conserved syntenic interval in the
human genome.
For 80% of mouse genomes, the best match in the human genome in turn has
its best match against that same mouse gene. These are termed 1:1 orthologues.
For less than 1% of the predicted mouse genes there was no homologous
predicted human gene.
Those genes that may seem to be mouse-specific may correspond to human genes
that are still missing due to the incompleteness of the human genome sequence.
De novo gene addition in the mouse lineage and gene deletion in the human
lineage have not significantly altered the gene repertoire.
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De novo gene prediction
dual-genome de novo gene predition in 2 steps
(1)
retain only multi-exon gene predictions for which there were corresponding
consecutive exons with an intron in an aligned position in both species.
(2)
require the presence of adjacent exons in both species
Authors expect about 1000 new gene predictions would be validated by RT-PCR.
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Mouse proteome
Taxonomic breakdown of homologues of mouse proteins according to
taxonomic range. Note that only a small fraction of genes are possibly
rodent-specific (<1%) as compared with those shared with other mammals
(14%, not rodent-specific); shared with chordates (6%, not mammalianspecific); shared with metazoans (27%, not chordate-specific); shared with
eukaryotes (29%, not metazoan-specific); and shared with prokaryotes and
other organisms (23%, not eukaryotic-specific).
The mouse genome. Nature 420, 520 - 562
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Gene ontology annotations
Gene ontology (GO) annotations for mouse and
human proteins. The GO terms assigned to mouse
(blue) and human (red) proteins based on sequence
matches to InterPro domains are grouped into
approximately a dozen categories. These categories
fell within each of the larger ontologies of cellular
component (a) molecular function (b) and biological
process (c) (D. Hill, personal communication). In
general, mouse has a similar percentage of proteins
compared with human in most categories. The
apparently significant difference between the number
of mouse and human proteins in the translational
apparatus category of the cellular component
ontology may be due to ribosomal protein
pseudogenes incorrectly assigned as genes in
mouse.
The mouse genome. Nature 420, 520 - 562
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Protein families
As expected, most of the
protein or domain
families have similar
sizes in human and
mouse.
Largest differences in
high mobility group
HMG1/2 and ubiquitin.
The mouse genome. Nature 420, 520 - 562
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Evolution of orthologues
two measures:
- percentage of amino acid identity
- KA / KS ratio
Orthologues generally have lower values for KA / KS e.g. < 0.05
because the proteins are subject to relatively strong purifying selection.
The mouse genome. Nature 420, 520 - 562
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Purifying selection
Domain prediction with SMART:
Domains are under greater purifying selection than regions not containing domains.
Consistent with hypothesis that domains are under greater structural and
functional constraints than unstructured, domain-free regions.
Also, domain families with enzymatic activitiy were found to have a lower KA / KS
ration than non-enzymatic domains.
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Summary
* the mouse genome is about 14% smaller than the human genome. The
difference probably reflects a higher rate of deletion in mouse.
* over 90% of the mouse and human genomes can be partitioned into
corresponding regions of conserved synteny (segments in which the gene order
in the most recent common ancestor has been conserved in both species)
* at the nucleotide level, ca. 40% of the human genome can be aligned to the
mouse genome. These sequences seem to represent most of the orthologous
sequences that remain in both lineages from the common ancestor. The rest
was probably deleted in one or both genomes.
* the neutral substitution rate has been roughly half a nucleotide substitution per
site since the divergence of the species. About twice as many of these
substitutions have occurred in mouse as in human.
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Summary
* the proportion of small (50-100 bp) segments in the mammalian genome that is
under (purifying) selection is ca. 5%, i.e. much higher than can be explained by
protein-coding sequences alone.
 genome contains many additional features (UTRs, regulatory elements, nonprotein-coding genes, chromosomal structural elements) under selection for
biological function!
* the mammalian genome is evolving in a non-uniform manner, various measures
of divergence showing substantial variation across the genome.
* mouse and human genomes each seem to contain ca. 30.000 protein-coding
genes. The proportion of mouse genes with a single identifiable orthologue in the
human genome is ca. 80%. The proportion of mouse genes without any homologue
currently detectable in the human genome (and vice versa) is < 1%.
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