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Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

Watson CT, Steinberg KM, Graves TA, Warren RL, Malig M, Schein J, Wilson RK, Holt RA, Eichler EE, Breden F - Genes Immun. (2014)

Bottom Line: The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap.Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters.Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada [2] Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

ABSTRACT
Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.

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Detection of putative sequence exchange event between IGK proximal and distal gene clusters(A) Pair-wise alignments between proximal and distal segmental duplications in the CH17 haplotype and NG_000834.1 (proximal) and NG_000833.1 (distal) assemblies (Abbreviations: Ka, NG_000834.1/NG_000833.1 assemblies; prox, proximal; dist, distal). The region where Ka-prox and Ka-dist show stronger similarity than CH17-dist and Ka-prox highlights a potential region of sequence exchange between the two NG_000834.1 and NG_000833.1 assemblies (red box). Coordinates (GRCh37) for the proximal cluster are shown on the X axis. (B) Top panel shows a four-way sequence alignment of a 22.5 kb region from the proximal and distal units from within the red box in (A). Blue tick marks indicate bp SNP differences between the sequences. Upward pointing red arrows indicate boundaries of regions where the NG_000833.1 distal sequence aligns with a higher sequence similarity to the NG_000834.1/NG_000833.1 assemblies and CH17 proximal sequences than to the CH17 distal sequence, indicative of exchange between proximal and distal regions of the NG_000834.1/NG_000833.1 assemblies (sequence similarities: Ka-dist/CH17-dist=98.7%; Ka-dist/Ka-prox=99.7%). A DSS recombination analysis ((70); see methods) using the same four-way sequence alignment is shown in the bottom panel. The two peaks with the strongest DSS values (downward pointing red arrows) correspond to the predicted breakpoints shown in the top panel based on sequence similarity values. The dotted line across the chart indicates the significance threshold based on the  distribution of DSS values calculated assuming no recombination.
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Figure 3: Detection of putative sequence exchange event between IGK proximal and distal gene clusters(A) Pair-wise alignments between proximal and distal segmental duplications in the CH17 haplotype and NG_000834.1 (proximal) and NG_000833.1 (distal) assemblies (Abbreviations: Ka, NG_000834.1/NG_000833.1 assemblies; prox, proximal; dist, distal). The region where Ka-prox and Ka-dist show stronger similarity than CH17-dist and Ka-prox highlights a potential region of sequence exchange between the two NG_000834.1 and NG_000833.1 assemblies (red box). Coordinates (GRCh37) for the proximal cluster are shown on the X axis. (B) Top panel shows a four-way sequence alignment of a 22.5 kb region from the proximal and distal units from within the red box in (A). Blue tick marks indicate bp SNP differences between the sequences. Upward pointing red arrows indicate boundaries of regions where the NG_000833.1 distal sequence aligns with a higher sequence similarity to the NG_000834.1/NG_000833.1 assemblies and CH17 proximal sequences than to the CH17 distal sequence, indicative of exchange between proximal and distal regions of the NG_000834.1/NG_000833.1 assemblies (sequence similarities: Ka-dist/CH17-dist=98.7%; Ka-dist/Ka-prox=99.7%). A DSS recombination analysis ((70); see methods) using the same four-way sequence alignment is shown in the bottom panel. The two peaks with the strongest DSS values (downward pointing red arrows) correspond to the predicted breakpoints shown in the top panel based on sequence similarity values. The dotted line across the chart indicates the significance threshold based on the distribution of DSS values calculated assuming no recombination.

Mentions: Previous analysis of sequence similarity between shared homology blocks of proximal and distal segmental duplication units, which comprise the majority of the IGK locus, revealed that over the majority of the locus these two clusters are >98% similar (23). Segmental duplications are known to facilitate sequence exchange via non-allelic homologous recombination and interlocus gene conversion (36, 37); however, given the lack of reference sequence data this has not been investigated in the IGK locus. To address this, we conducted pair-wise comparisons of distal and proximal regions from CH17 and NG_000834.1/NG_000833.1 assemblies to search for large tracts of shared sequence (Figure 3A). The expectation is that sequences should be most similar between homologous regions of the NG_000834.1/NG_000833.1 and CH17 assemblies, whereas higher similarity between proximal and distal regions within a given assembly would suggest the potential occurrence of sequence exchange. Using this approach, we identified a large ~16.7 kb region that showed higher identity between the proximal and distal units of the NG_000834.1/NG_000833.1 assemblies than between the CH17 distal and NG_000834.1 IGK proximal units (Figure 3A). This region included two IGKV genes for which we observed allelic variants between the NG_000834.1/NG_000833.1 assemblies and CH17 haplotypes. Four-way sequence alignments of this region show that the CH17 distal unit was most unique compared to the other three sequences (Figure 3B), providing evidence that distal and proximal units have undergone sequence exchange at some point in the past, making the distal unit more similar to the proximal unit in this region represented in the NG_000834.1/NG_000833.1 assemblies. It is important to note, however, that the distal fragment of the initial assembly generated by Kawasaki et al. (23) (NG_000833.1) harbors many unique bp differences compared to the other three sequences (blue tick marks, Figure 3B), which could be suggestive of the occurrence of mutation following the predicted sequence exchange event. Further analysis of this multi-sequence alignment using the Difference of Sums of Squares (DSS) method for recombination detection also predicted two potential flanking recombination breakpoints within the expected regions based on visual inspection of the sequence alignment and comparison of sequence similarities. We also analyzed sequence from two BAC clones in the proximal and distal clusters from the RPCI-11 BAC library (24); providing further support for this predicted event, this analysis revealed that these carried the same variants observed in the NG_000834.1/NG_000833.1 assemblies. An alternative explanation, although less parsimonious given the data presented, could be that this region of the IGKV distal cluster in the CH17 haplotype has undergone more rapid sequence divergence since the original duplication of the IGKV distal and proximal segments. The sequencing of additional haplotypes in the distal and proximal IGKV clusters will likely provide more insight into the mechanisms underlying these observed sequence signatures.


Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

Watson CT, Steinberg KM, Graves TA, Warren RL, Malig M, Schein J, Wilson RK, Holt RA, Eichler EE, Breden F - Genes Immun. (2014)

Detection of putative sequence exchange event between IGK proximal and distal gene clusters(A) Pair-wise alignments between proximal and distal segmental duplications in the CH17 haplotype and NG_000834.1 (proximal) and NG_000833.1 (distal) assemblies (Abbreviations: Ka, NG_000834.1/NG_000833.1 assemblies; prox, proximal; dist, distal). The region where Ka-prox and Ka-dist show stronger similarity than CH17-dist and Ka-prox highlights a potential region of sequence exchange between the two NG_000834.1 and NG_000833.1 assemblies (red box). Coordinates (GRCh37) for the proximal cluster are shown on the X axis. (B) Top panel shows a four-way sequence alignment of a 22.5 kb region from the proximal and distal units from within the red box in (A). Blue tick marks indicate bp SNP differences between the sequences. Upward pointing red arrows indicate boundaries of regions where the NG_000833.1 distal sequence aligns with a higher sequence similarity to the NG_000834.1/NG_000833.1 assemblies and CH17 proximal sequences than to the CH17 distal sequence, indicative of exchange between proximal and distal regions of the NG_000834.1/NG_000833.1 assemblies (sequence similarities: Ka-dist/CH17-dist=98.7%; Ka-dist/Ka-prox=99.7%). A DSS recombination analysis ((70); see methods) using the same four-way sequence alignment is shown in the bottom panel. The two peaks with the strongest DSS values (downward pointing red arrows) correspond to the predicted breakpoints shown in the top panel based on sequence similarity values. The dotted line across the chart indicates the significance threshold based on the  distribution of DSS values calculated assuming no recombination.
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Related In: Results  -  Collection

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Figure 3: Detection of putative sequence exchange event between IGK proximal and distal gene clusters(A) Pair-wise alignments between proximal and distal segmental duplications in the CH17 haplotype and NG_000834.1 (proximal) and NG_000833.1 (distal) assemblies (Abbreviations: Ka, NG_000834.1/NG_000833.1 assemblies; prox, proximal; dist, distal). The region where Ka-prox and Ka-dist show stronger similarity than CH17-dist and Ka-prox highlights a potential region of sequence exchange between the two NG_000834.1 and NG_000833.1 assemblies (red box). Coordinates (GRCh37) for the proximal cluster are shown on the X axis. (B) Top panel shows a four-way sequence alignment of a 22.5 kb region from the proximal and distal units from within the red box in (A). Blue tick marks indicate bp SNP differences between the sequences. Upward pointing red arrows indicate boundaries of regions where the NG_000833.1 distal sequence aligns with a higher sequence similarity to the NG_000834.1/NG_000833.1 assemblies and CH17 proximal sequences than to the CH17 distal sequence, indicative of exchange between proximal and distal regions of the NG_000834.1/NG_000833.1 assemblies (sequence similarities: Ka-dist/CH17-dist=98.7%; Ka-dist/Ka-prox=99.7%). A DSS recombination analysis ((70); see methods) using the same four-way sequence alignment is shown in the bottom panel. The two peaks with the strongest DSS values (downward pointing red arrows) correspond to the predicted breakpoints shown in the top panel based on sequence similarity values. The dotted line across the chart indicates the significance threshold based on the distribution of DSS values calculated assuming no recombination.
Mentions: Previous analysis of sequence similarity between shared homology blocks of proximal and distal segmental duplication units, which comprise the majority of the IGK locus, revealed that over the majority of the locus these two clusters are >98% similar (23). Segmental duplications are known to facilitate sequence exchange via non-allelic homologous recombination and interlocus gene conversion (36, 37); however, given the lack of reference sequence data this has not been investigated in the IGK locus. To address this, we conducted pair-wise comparisons of distal and proximal regions from CH17 and NG_000834.1/NG_000833.1 assemblies to search for large tracts of shared sequence (Figure 3A). The expectation is that sequences should be most similar between homologous regions of the NG_000834.1/NG_000833.1 and CH17 assemblies, whereas higher similarity between proximal and distal regions within a given assembly would suggest the potential occurrence of sequence exchange. Using this approach, we identified a large ~16.7 kb region that showed higher identity between the proximal and distal units of the NG_000834.1/NG_000833.1 assemblies than between the CH17 distal and NG_000834.1 IGK proximal units (Figure 3A). This region included two IGKV genes for which we observed allelic variants between the NG_000834.1/NG_000833.1 assemblies and CH17 haplotypes. Four-way sequence alignments of this region show that the CH17 distal unit was most unique compared to the other three sequences (Figure 3B), providing evidence that distal and proximal units have undergone sequence exchange at some point in the past, making the distal unit more similar to the proximal unit in this region represented in the NG_000834.1/NG_000833.1 assemblies. It is important to note, however, that the distal fragment of the initial assembly generated by Kawasaki et al. (23) (NG_000833.1) harbors many unique bp differences compared to the other three sequences (blue tick marks, Figure 3B), which could be suggestive of the occurrence of mutation following the predicted sequence exchange event. Further analysis of this multi-sequence alignment using the Difference of Sums of Squares (DSS) method for recombination detection also predicted two potential flanking recombination breakpoints within the expected regions based on visual inspection of the sequence alignment and comparison of sequence similarities. We also analyzed sequence from two BAC clones in the proximal and distal clusters from the RPCI-11 BAC library (24); providing further support for this predicted event, this analysis revealed that these carried the same variants observed in the NG_000834.1/NG_000833.1 assemblies. An alternative explanation, although less parsimonious given the data presented, could be that this region of the IGKV distal cluster in the CH17 haplotype has undergone more rapid sequence divergence since the original duplication of the IGKV distal and proximal segments. The sequencing of additional haplotypes in the distal and proximal IGKV clusters will likely provide more insight into the mechanisms underlying these observed sequence signatures.

Bottom Line: The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap.Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters.Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada [2] Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

ABSTRACT
Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH.

Show MeSH
Related in: MedlinePlus