Distribution Of Indels Research Articles

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

THU209 Small INDELs In The Androgen Receptor Gene: Phenotype Implications And Mechanisms Of Mutagenesis In Androgen Insensitivity Syndrome

Abstract Disclosure: R.L. Batista: None. R.M. Ramos: None. A.F. Afonso: None. R.J. Petroli: None. M.Y. Nishi: None. M.M. Ferrari: None. S. Domenice: None. B.B. Mendonca: None. Small indels are insertions or deletions of one or more nucleotides into the DNA sequence. Indels are highly abundant in human genomes and have contributed massively to genome evolution. However, the role of indels and their underlined mechanisms of mutagenesis in Mendelian disorders still needs to be appreciated. To explore the contribution of indels in a monogenic disorder, we analyzed all indels in the androgen receptor gene, including three novel indels, using Androgen Insensitivity Syndrome (AIS) as a model. The goal was to profile indels in AIS. To build this profile, we analyzed the indel distribution through the AR coding region, compared with genomic and exonic populational data, and analyzed their impact on the AIS phenotype and their likely mechanism of mutagenesis. We present 82 different indels in the AR gene reported in individuals with AIS. In most cases (n=78; 95.1%), the phenotype is complete AIS (CAIS). There are four AR indels reported in mild AIS. Most short indels in the AR gene among AIS are frameshift indels (n=64; 79%). Indels are distributed along the entire AR gene, with NTD domain predominance (n=45; 54.9%), followed by LTD domain (n=28; 34.1%). Most are small deletions (n=49; 59.7%), followed by small insertions (n=29; 35.3%) and complex frameshift indels (n=4; 4.8%). Most indels are short (<10 bp; n=81 = 98.8%), with an average of 2.4 nucleotides by indel. Most indels are at non-repetitive sequences with no changes in copy counting (n = 68; 82.9%). Thirteen indels were identified within tandem repeats (15.8%), whereas seven indels were at homopolymers run (8.5%). Trinucleotide regions (Poly-Q, Poly-G) are prone to indels, but indels also happen in other trinucleotide stretches (Poli-L, Poly-P). Regarding the change in copy counting, thirty-nine (47.5%) indels are CCC indels. Indel reported in MAIS is all located at tandem repeats, while all at non-repetitive sequences caused CAIS. 3.65% of AR indels are at a 6bp consensus sequence (TG A/G A/G G/T A/C). In summary, indels represent a significant portion of the variants identified in the AR gene in patients with AIS. Unlike the non-synonymous variants related to this gene, there is a genotype-phenotype correlation (indel - CAIS congruency). However, small NFS indels at small triplet runs can slightly compromise AR function leading to the mild phenotype. Homopolymer runs, and trinucleotide repeats are exonic regions at risk for indels. Slippage explains half of the cases of AR indels. These results increase the molecular understanding of the androgen receptor disruption and the underlined mechanisms of indel mutagenesis in exonic regions and their implication on phenotype. Presentation: Thursday, June 15, 2023

DeltaMSI: artificial intelligence-based modeling of microsatellite instability scoring on next-generation sequencing data

BackgroundDNA mismatch repair deficiency (dMMR) testing is crucial for detection of microsatellite unstable (MSI) tumors. MSI is detected by aberrant indel length distributions of microsatellite markers, either by visual inspection of PCR-fragment length profiles or by automated bioinformatic scoring on next-generation sequencing (NGS) data. The former is time-consuming and low-throughput while the latter typically relies on simplified binary scoring of a single parameter of the indel distribution. The purpose of this study was to use machine learning to process the full complexity of indel distributions and integrate it into a robust script for screening of dMMR on small gene panel-based NGS data of clinical tumor samples without paired normal tissue.MethodsScikit-learn was used to train 7 models on normalized read depth data of 36 microsatellite loci in a cohort of 133 MMR proficient (pMMR) and 46 dMMR tumor samples, taking loss of MLH1/MSH2/PMS2/MSH6 protein expression as reference method. After selection of the optimal model and microsatellite panel the two top-performing models per locus (logistic regression and support vector machine) were integrated into a novel script (DeltaMSI) for combined prediction of MSI status on 28 marker loci at sample level. Diagnostic performance of DeltaMSI was compared to that of mSINGS, a widely used script for MSI detection on unpaired tumor samples. The robustness of DeltaMSI was evaluated on 1072 unselected, consecutive solid tumor samples in a real-world setting sequenced using capture chemistry, and 116 solid tumor samples sequenced by amplicon chemistry. Likelihood ratios were used to select result intervals with clinical validity.ResultsDeltaMSI achieved higher robustness at equal diagnostic power (AUC = 0.950; 95% CI 0.910–0.975) as compared to mSINGS (AUC = 0.876; 95% CI 0.823–0.918). Its sensitivity of 90% at 100% specificity indicated its clinical potential for high-throughput MSI screening in all tumor types.Clinical Trial Number/IRB B1172020000040, Ethical Committee, AZ Delta General Hospital.

Conservation of an Agrobacterium cT-DNA insert in Camellia section Thea reveals the ancient origin of tea plants from a genetically modified ancestor.

Many higher plants contain cellular T-DNA (cT-DNA) sequences from Agrobacterium and have been called "natural genetically modified organisms" (nGMOs). Among these natural transformants, the tea plant Camellia sinensis var. sinensis cv. Shuchazao contains a single 5.5 kb T-DNA fragment (CaTA) with three inactive T-DNA genes, with a 1 kb inverted repeat at the ends. Camellia plants are allogamous, so that each individual may contain two different CaTA alleles. 142 Camellia accessions, belonging to 10 of 11 species of the section Thea, were investigated for the presence of CaTA alleles. All accessions were found to contain the CaTA insert, showing that section Thea derives from a single transformed ancestor. Allele phasing showed that 82 accessions each contained two different CaTA alleles, 60 others had a unique allele. A phylogenetic tree of these 225 alleles showed two separate groups, A and B, further divided into subgroups. Indel distribution corresponded in most cases with these groups. The alleles of the different Camellia species were distributed over groups A and B, and different species showed very similar CaTA alleles. This indicates that the species boundaries for section Thea may not be precise and require revision. The nucleotide divergence of the indirect CaTA repeats indicates that the cT-DNA insertion took place about 15 Mio years ago, before the emergence of section Thea. The CaTA structure of a C. fangchengensis accession has an exceptional structure. We present a working model for the origin and evolution of nGMO plants derived from allogamous transformants.

Investigation of genome-wide InDel distribution and segregation in Populus with restriction-site associated DNA sequencing data

Insertion or deletion polymorphism (InDel) is one of the main genetic variations in plant genomes. However, there are few studies on InDels across the whole genome in Populus. In this study, we investigated genome-wide InDels in Populus deltoides and Populus simonii and InDel segregation in their F1 hybrid population with restriction-site associated DNA sequencing (RAD-seq) data. A total of 119,066 InDels were identified in P. deltoides and P. simonii according to the reference genome of Populus trichocarpa, including 58,532 unique InDels in P. deltoides, 54,469 unique InDels in P. simonii, and 6,065 common InDels in both. Meanwhile, the distribution of these InDels was analyzed along chromosomes, indicating that the distribution patterns for both species were largely similar, but the average InDel density was slightly higher in P. deltoides than in P. simonii. GO annotation and enrichment analysis of those genes harboring InDels showed the same patterns between the two poplar species. It is interesting to find that the ratio (~ 46%) of the common InDels within genes to all common InDels was higher than that of the InDels within genes to all InDels in P. deltoides (~ 35%) or in P. simonii (~ 34%), possibly indicating that those InDels are more conservative between poplar species. Moreover, investigation of the InDel segregation patterns demonstrated that a large number of Mendelian InDels could be selected for genetic mapping in the F1 hybrid population. RAD-seq provides genome-wide insights into the InDel distributions in P. deltoides and P. simonii and the segregation patterns in their progeny, providing valuable genomic variation information for genetic and evolutionary studies in Populus.

Combined +58 and +55 BCL11A enhancer Editing Yields Exceptional Efficiency, Specificity and HbF Induction in Human and NHP Preclinical Models

Targeting the BCL11A erythroid enhancer by gene editing is a promising approach to fetal hemoglobin induction for beta-hemoglobinopathies. HbF levels vary widely among individuals, suggesting potential heterogeneity in HbF responses after therapeutic intervention. We hypothesize that maximizing both gene edit frequency and HbF induction potential could promote consistently favorable clinical outcomes. Here we compared CRISPR-Cas9 endonuclease editing of the BCL11A +58 enhancer with alternative gene modification approaches, including +55 erythroid enhancer editing alone or in combination with the +58 enhancer, as well as editing targeting the HBG1/2 promoter -115 BCL11A binding site and transduction by an shRNA knocking down the BCL11A transcript in erythroid precursors. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in the most potent HbF induction (52.4%±6.3%) of tested approaches (BCL11A +58 editing alone, 29.1%±3.9%; BCL11A +55 editing alone, 34.8±5.1%; HBG1/2 promoter editing, 34.1% ±5.4%; shmiR-BCL11A, 32.2%±4.4%; mock, 7.6%±3.4%). Based on assays in bulk and single cell derived erythroid cultures and xenografted immunodeficient mice, we found that disruption of core half E-box/GATA motifs at both the +58 and +55 enhancers was associated with greatest HbF induction, whether by small indels, interstitial 3.1 kb deletion, or 3.1 kb inversion. Rare gene edited clones with alleles that only partially disrupted these motifs were associated with intermediate HbF induction phenotypes. Combined editing of BCL11A +58 and +55 enhancers was compatible with HSC self-renewal in primary and secondary xenotransplant, with intact lymphoid, myeloid and erythroid repopulation. We conducted gene-edited cell product manufacturing process development and developed conditions using a MaxCyte electroporation instrument achieving mean 97.3±1.8% gene edits and 88.9%±6.4% viability 24 hours after electroporation in 3 engineering runs at clinical scale. We obtained similar results at small-scale with plerixafor-mobilized HSPCs from sickle cell disease (SCD) donors or G-CSF mobilized PBMCs from transfusion-dependent beta-thalassemia (TDT) donors, including 94.2%±4.4%, 99.5%±0.3% and 91.8%±6.3% of gene edits in engrafting cells from NBSGW 16 week mouse bone marrow of healthy, SCD and TDT donors respectively. Off-target analyses by pooled amplicon sequencing of 601 candidate off-target sites for the +58 and +55 targeting sgRNAs, nominated by a range of computational (CRISPRme) and experimental (GUIDE-seq and ONE-seq) methods, did not identify reference genome off-target edits at a sensitivity of 0.1% allele frequency. We evaluated +58/+55 enhancer combined targeting in nonhuman primates by performing ribonucleoprotein (RNP) electroporation in rhesus macaque mobilized peripheral blood CD34+ HSPCs with autologous re-infusion following busulfan myeloablation. We observed highly efficient gene edit frequency (85.2%, 88.8% and 84.9%) and durable HbF induction (26.4%, 57.5%, and 45.9% F-cells and 12.7%, 41.9%, and 28% gamma-globin) in the peripheral blood in 3 animals at most recent recorded time point post infusion (127, 78, and 54 weeks respectively). Single colony analyses and bulk ddPCR and unidirectional sequencing demonstrated that the long-term engrafting cells displayed a similar distribution of indels, 3.1 kb deletions, and 3.1 kb inversions as the input cell products. Erythroid stress due to hydroxyurea treatment, with or without phlebotomy, was associated with substantially augmented HbF responses (to 75.9%, 88.2%, and 57.8% F-cells and 47.9%, 68%, and 35.7% gamma-globin). No hematologic or other toxicities attributable to gene editing were observed.Together these results suggest that combined BCL11A +58 and +55 erythroid enhancer editing produces highly efficient on-target allelic disruption, erythroid-specific BCL11A downregulation, heightened HbF induction capacity compared to alternative approaches, preserved long-term multilineage engraftment potential by both human xenotransplant and rhesus autotransplant assays, and absence of evident genotoxicity, under clinically relevant SpCas9 RNP electroporation conditions. DisclosuresNo relevant conflicts of interest to declare.

Phylogenetic measures of indel rate variation among the HIV-1 group M subtypes

The transmission fitness and pathogenesis of HIV-1 is disproportionately influenced by evolution in the five variable regions (V1–V5) of the surface envelope glycoprotein (gp120). Insertions and deletions (indels) are a significant source of evolutionary change in these regions. However, the rate and composition of indels has not yet been quantified through a large-scale comparative analysis of HIV-1 sequences. Here, we develop and report results from a phylogenetic method to estimate indel rates for the gp120 variable regions across five major subtypes and two circulating recombinant forms (CRFs) of HIV-1 group M. We processed over 26,000 published HIV-1 gp120 sequences, from which we extracted 6,605 sequences for phylogenetic analysis. We reconstructed time-scaled phylogenies by maximum likelihood and fit a binomial-Poisson model to the observed distribution of indels between closely related pairs of sequences in each tree (cherries). By focusing on cherries in each tree, we obtained phylogenetically independent indel reconstructions, and the shorter time scales in cherries reduced the bias due to purifying selection. Rate estimates ranged from to indels/nt/year and varied significantly among variable regions and subtypes. Indel rates were significantly lower in V3 relative to V1, and were also lower in HIV-1 subtype B relative to the 01_AE reference. We also found that V1, V2, and V4 tended to accumulate significantly longer indels. Furthermore, we observed that the nucleotide composition of indels was distinct from the flanking sequence, with higher frequencies of G and lower frequencies of T. Indels affected N-linked glycosylation sites more often in V1 and V2 than expected by chance, consistent with positive selection on glycosylation patterns within these regions. These results represent the first comprehensive measures of indel rates in HIV-1 gp120 across multiple subtypes and CRFs, and identifies novel and unexpected patterns for further research in the molecular evolution of HIV-1.

Whole-genome sequencing reveals mutational landscape underlying phenotypic differences between two widespread Chinese cattle breeds.

Whole-genome sequencing provides a powerful tool to obtain more genetic variability that could produce a range of benefits for cattle breeding industry. Nanyang (Bos indicus) and Qinchuan (Bos taurus) are two important Chinese indigenous cattle breeds with distinct phenotypes. To identify the genetic characteristics responsible for variation in phenotypes between the two breeds, in the present study, we for the first time sequenced the genomes of four Nanyang and four Qinchuan cattle with 10 to 12 fold on average of 97.86% and 98.98% coverage of genomes, respectively. Comparison with the Bos_taurus_UMD_3.1 reference assembly yielded 9,010,096 SNPs for Nanyang, and 6,965,062 for Qinchuan cattle, 51% and 29% of which were novel SNPs, respectively. A total of 154,934 and 115,032 small indels (1 to 3 bp) were found in the Nanyang and Qinchuan genomes, respectively. The SNP and indel distribution revealed that Nanyang showed a genetically high diversity as compared to Qinchuan cattle. Furthermore, a total of 2,907 putative cases of copy number variation (CNV) were identified by aligning Nanyang to Qinchuan genome, 783 of which (27%) encompassed the coding regions of 495 functional genes. The gene ontology (GO) analysis revealed that many CNV genes were enriched in the immune system and environment adaptability. Among several CNV genes related to lipid transport and fat metabolism, Lepin receptor gene (LEPR) overlapping with CNV_1815 showed remarkably higher copy number in Qinchuan than Nanyang (log2 (ratio) = -2.34988; P value = 1.53E-102). Further qPCR and association analysis investigated that the copy number of the LEPR gene presented positive correlations with transcriptional expression and phenotypic traits, suggesting the LEPR CNV may contribute to the higher fat deposition in muscles of Qinchuan cattle. Our findings provide evidence that the distinct phenotypes of Nanyang and Qinchuan breeds may be due to the different genetic variations including SNPs, indels and CNV.

Chloroplast DNA Structural Variation, Phylogeny, and Age of Divergence among Diploid Cotton Species.

The cotton genus (Gossypium spp.) contains 8 monophyletic diploid genome groups (A, B, C, D, E, F, G, K) and a single allotetraploid clade (AD). To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome in this group, we performed a comparative analysis of 19 Gossypium chloroplast genomes, six reported here for the first time. Nucleotide distance in non-coding regions was about three times that of coding regions. As expected, distances were smaller within than among genome groups. Phylogenetic topologies based on nucleotide and indel data support for the resolution of the 8 genome groups into 6 clades. Phylogenetic analysis of indel distribution among the 19 genomes demonstrates contrasting evolutionary dynamics in different clades, with a parallel genome downsizing in two genome groups and a biased accumulation of insertions in the clade containing the cultivated cottons leading to large (for Gossypium) chloroplast genomes. Divergence time estimates derived from the cpDNA sequence suggest that the major diploid clades had diverged approximately 10 to 11 million years ago. The complete nucleotide sequences of 6 cpDNA genomes are provided, offering a resource for cytonuclear studies in Gossypium.

Genome-wide identification of novel genetic markers from RNA sequencing assembly of diverse Aegilops tauschii accessions.

The wild species in the Triticeae tribe are tremendous resources for crop breeding due to their abundant natural variation. However, their huge and highly repetitive genomes have hindered the establishment of physical maps and the completeness of their genome sequences. To develop molecular markers for the efficient utilization of their valuable traits while avoiding their genome complexity, we assembled RNA sequences of ten representative accessions of Aegilops tauschii, the progenitor of the wheat D genome, and estimated single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). The deduced unigenes were anchored to the chromosomes of Ae. tauschii and barley. The SNPs and indels in the anchored unigenes, covering entire chromosomes, were sufficient for linkage map construction, even in combinations between the genetically closest accessions. Interestingly, the resolution of SNP and indel distribution on barley chromosomes was slightly higher than on Ae. tauschii chromosomes. Since barley chromosomes are regarded as virtual chromosomes of Triticeae species, our strategy allows capture of genetic markers arranged on the chromosomes in order based on the conserved synteny. The resolution of these genetic markers will be comparable to that of the Ae. tauschii whose draft genome sequence is available. Our procedure should be applicable to marker development for Triticeae species, which have no draft sequences available.

Insertion-deletions burden in copy number polymorphisms of the Tibetan population.

BACKGROUND:Many studies have been conducted to identify either insertions-deletions (inDels) or copy number variations (CNVs) in humans, but few studies have been conducted to identify both of these forms coexisting in the same region.AIMS AND OBJECTIVES:To map the functionally significant sites within human genes that are likely to influence human traits and diseases.MATERIALS AND METHODS:In this report, we describe an inDel map in the 1051 Tibetan CNV regions obtained through CNV genotyping using Affymetrix Genome-wide single nucleotide polymorphism 6.0 chip. InDel polymorphisms in these copy number polymorphism regions were identified with a computational approach using the 2500 deoxyribonucleic acid sequences obtained from the 1000 Genome Project.RESULTS:The study identified a total of 95935 inDels that range from 1 bp to several bps in length which were found scattered across regulatory regions, exons and in introns of genes underlying the CNVs. A study on the distribution of inDels revealed that the majority of inDels were found in coding regions of the genome than the noncoding, while within the genes, inDels in intron regions were more followed by exonic regions and finally the regulatory regions.CONCLUSION:Study of inDels in CNV regions contribute to the enhanced understanding of the role played by the two variations and their collective influence on the genome. Further, a collection of these inDel genetic markers will aid in genetic mapping, further understanding of the phenotypic variability, identification of disease genes and in detecting novel CNVs.

Systematic Analysis of microRNA Targeting Impacted by Small Insertions and Deletions in Human Genome

MicroRNAs (miRNAs) are small noncoding RNA that play an important role in posttranscriptional regulation of mRNA. Genetic variations in miRNAs or their target sites have been shown to alter miRNA function and have been associated with risk for several diseases. Previous studies have focused on the most abundant type of genetic variations, single nucleotide polymorphisms (SNPs) that affect miRNA-mRNA interactions. Here, we systematically identified small insertions and deletions (indels) in miRNAs and their target sites, and investigated the effects of indels on miRNA targeting. We studied the distribution of indels in miRNAs and their target sites and found that indels in mature miRNAs, experimentally supported miRNA target sites and PAR-CLIP footprints have significantly lower density compared to flanking regions. We identified over 20 indels in the seed regions of miRNAs, which may disrupt the interactions between these miRNAs and their target genes. We also identified hundreds of indels that alter experimentally supported miRNA target sites. We mapped these genes to human disease pathways to identify indels that affect miRNA targeting in these pathways. We also used the results of genome-wide association studies (GWAS) to identify potential links between miRNA-related indels and diseases.

The complete chloroplast DNA sequence of Eleutherococcus senticosus (Araliaceae); comparative evolutionary analyses with other three asterids.

This study reports the complete chloroplast (cp) DNA sequence of Eleutherococcus senticosus (GenBank: JN 637765), an endangered endemic species. The genome is 156,768 bp in length, and contains a pair of inverted repeat (IR) regions of 25,930 bp each, a large single copy (LSC) region of 86,755 bp and a small single copy (SSC) region of 18,153 bp. The structural organization, gene and intron contents, gene order, AT content, codon usage, and transcription units of the E. senticosus chloroplast genome are similar to that of typical land plant cp DNA. We aligned and analyzed the sequences of 86 coding genes, 19 introns and 113 intergenic spacers (IGS) in three different taxonomic hierarchies; Eleutherococcus vs. Panax, Eleutherococcus vs. Daucus, and Eleutherococcus vs. Nicotiana. The distribution of indels, the number of polymorphic sites and nucleotide diversity indicate that positional constraint is more important than functional constraint for the evolution of cp genome sequences in Asterids. For example, the intron sequences in the LSC region exhibited base substitution rates 5-11-times higher than that of the IR regions, while the intron sequences in the SSC region evolved 7-14-times faster than those in the IR region. Furthermore, the Ka/Ks ratio of the gene coding sequences supports a stronger evolutionary constraint in the IR region than in the LSC or SSC regions. Therefore, our data suggest that selective sweeps by base collection mechanisms more frequently eliminate polymorphisms in the IR region than in other regions. Chloroplast genome regions that have high levels of base substitutions also show higher incidences of indels. Thirty-five simple sequence repeat (SSR) loci were identified in the Eleutherococcus chloroplast genome. Of these, 27 are homopolymers, while six are di-polymers and two are tri-polymers. In addition to the SSR loci, we also identified 18 medium size repeat units ranging from 22 to 79 bp, 11 of which are distributed in the IGS or intron regions. These medium size repeats may contribute to developing a cp genome-specific gene introduction vector because the region may use for specific recombination sites.

Reconstruction of structural evolution in the trnL intron P6b loop of symbiotic Nostoc (Cyanobacteria)

In this study we reconstruct the structural evolution of the hyper-variable P6b region of the group I trnLeu intron in a monophyletic group of lichen-symbiotic Nostoc strains and establish it as a useful marker in the phylogenetic analysis of these organisms. The studied cyanobacteria occur as photosynthetic and/or nitrogen-fixing symbionts in lichen species of the diverse Nephroma guild. Phylogenetic analyses and secondary structure reconstructions are used to improve the understanding of the replication mechanisms in the P6b stem-loop and to explain the observed distribution patterns of indels. The variants of the P6b region in the Nostoc clade studied consist of different combinations of five sequence modules. The distribution of indels together with the ancestral character reconstruction performed enables the interpretation of the evolution of each sequence module. Our results indicate that the indel events are usually associated with single nucleotide changes in the P6b region and have occurred several times independently. In spite of their homoplasy, they provide phylogenetic information for closely related taxa. Thus we recognize that features of the P6b region can be used as molecular markers for species identification and phylogenetic studies involving symbiotic Nostoc cyanobacteria.

Exome Sequencing of the t(4;14) and t(11;14) Translocation Specific Subgroups of MM

Abstract 1817The two most frequent etiological translocations in multiple myeloma (MM) are t(4;14), which deregulates FGFR3 and MMSET and has a poor outcome and t(11;14) which directly deregulates cyclin D1 and has an indolent course. The t(11;14) is present at 10–15% in both MGUS and MM but the t(4;14) is in only 3–4% of MGUS compared to 11% in MM. Consequently it is thought that patients with a t(4;14) have a less stable disease which progresses more quickly to myeloma than other subtypes. In order to address the hypothesis that cases with the t(4;14) are more prone to acquire mutations and so progress, we have compared the number and mutational spectrum of cases with these two variants.DNA was extracted from CD138-selected bone marrow cells from 10 t(4;14) and 12 t(11;14) cases of newly presenting MM. 50 ng of genomic DNA was used to capture the exome using the SureSelect Human All Exon 50Mb target enrichment set (Agilent). We have previously validated this approach and shown it to have parity with approaches using larger starting amounts of DNA. Libraries were prepared from tumor and non-tumor DNA from the same patient and sequenced using 76 bp paired end reads on a GAIIx (Illumina). Samples were sequenced to a median depth of 61x, with 99% >1x and 85% >20x exomic coverage. Following base calling and quality control metrics the raw fastq reads were aligned to the reference human genome. The Genome Analysis Tool Kit was used to call indels and single nucleotide variants (SNVs), with BreakDancer used to detect structural variants. These variant calls were recalibrated and soft filters applied to remove potential false-positives using dbSNP, HapMap and the thousand genomes project as truth sets. Variants that occurred in both the normal and tumor samples were filtered out and the tumor-specific variants were annotated using Annovar. As well as the identification of commonly affected genes, functional annotation enrichment analysis was used to identify commonly affected pathways.The group of 22 cases sequenced at the exome level showed a mutation spectrum that comprised 32,000 SNVs and 1,800 indels per patient, with 1,600 SNVs and 500 indels in the tumor sample only. Structural and copy number variants inferred from this data were also identified and validated previous results using other technologies. We identified 250 SNVs and indels, per patient, that were not in dbSNP and constitute tumor-acquired mutations. We were able to validate some of these mutations that we had previously analyzed using other platforms (98% concordance). The Ti/Tv ratio of mutations was not consistent with any specific exposure or mechanism. The distribution of indels was biased toward insertions rather than deletions, with both predominantly being multiples of three to produce in-frame mutants. In total sequencing data from 60 exomes is available and pathway analysis of the SNVs newly identified confirmed the deregulation of pathways previously identified as being mutated in myeloma, in addition we also identify novel deregulated genes and pathways.We note a consistent increase in the number of variants in the t(4;14). Each tumor had on average 60 non-synonymous SNVs per sample with a range of 29 to 101, some patients being clear outliers. There was a bias to an excess number of mutations within the t(4;14) group which did not reach statistical significance. Importantly, the overlap between the SNVs in individual patients was limited with few consistently mutated genes across the sample set as a whole. In contrast, pathway analysis of the genes mutated in these two different entities shows marked similarities, with more frequent involvement of genes mediating cell adhesion in the t(4;14)s. Although the t(4;14) group had a greater number of mutations, a larger number of genes were affected in the t(11;14) group with the number of mutated genes in two or more samples being 111 versus 237, respectively. This observation implies a more consistent group of genes are deregulated in the t(4;14) group, suggesting that they are under greater selective pressure than in the t(11;14) group.In this work we show a higher mutation frequency but with more limited numbers of genes affected in the t(4;14) group compared to the t(11;14) group. Overall, the data are consistent within the two etiologically distinct groups of MM having a similar spectrum of mutations driving disease progression, with a focus on pathway deregulation rather than any single gene. Disclosures:No relevant conflicts of interest to declare.

PhyloSim - Monte Carlo simulation of sequence evolution in the R statistical computing environment.

BackgroundThe Monte Carlo simulation of sequence evolution is routinely used to assess the performance of phylogenetic inference methods and sequence alignment algorithms. Progress in the field of molecular evolution fuels the need for more realistic and hence more complex simulations, adapted to particular situations, yet current software makes unreasonable assumptions such as homogeneous substitution dynamics or a uniform distribution of indels across the simulated sequences. This calls for an extensible simulation framework written in a high-level functional language, offering new functionality and making it easy to incorporate further complexity.ResultsPhyloSim is an extensible framework for the Monte Carlo simulation of sequence evolution, written in R, using the Gillespie algorithm to integrate the actions of many concurrent processes such as substitutions, insertions and deletions. Uniquely among sequence simulation tools, PhyloSim can simulate arbitrarily complex patterns of rate variation and multiple indel processes, and allows for the incorporation of selective constraints on indel events. User-defined complex patterns of mutation and selection can be easily integrated into simulations, allowing PhyloSim to be adapted to specific needs.ConclusionsClose integration with R and the wide range of features implemented offer unmatched flexibility, making it possible to simulate sequence evolution under a wide range of realistic settings. We believe that PhyloSim will be useful to future studies involving simulated alignments.

Important role of indels in somatic mutations of human cancer genes.

BackgroundCancer is clonal proliferation that arises owing to mutations in a subset of genes that confer growth advantage. More and more cancer related genes are found to have accumulated somatic mutations. However, little has been reported about mutational patterns of insertions/deletions (indels) in these genes.ResultsWe analyzed indels' abundance and distribution, the relative ratio between indels and somatic base substitutions and the association between those two forms of mutations in a large number of somatic mutations in the Catalogue of Somatic Mutations in Cancer database. We found a strong correlation between indels and base substitutions in cancer-related genes and showed that they tend to concentrate at the same locus in the coding sequences within the same samples. More importantly, a much higher proportion of indels were observed in somatic mutations, as compared to meiotic ones. Furthermore, our analysis demonstrated a great diversity of indels at some loci of cancer-related genes. Particularly in the genes with abundant mutations, the proportion of 3n indels in oncogenes is 7.9 times higher than that in tumor suppressor genes.ConclusionsThere are three distinct patterns of indel distribution in somatic mutations: high proportion, great abundance and non-random distribution. Because of the great influence of indels on gene function (e.g., the effect of frameshift mutation), these patterns indicate that indels are frequently under positive selection and can often be the 'driver mutations' in oncogenesis. Such driver forces can better explain why much less frameshift mutations are in oncogenes while much more in tumor suppressor genes, because of their different function in oncogenesis. These findings contribute to our understanding of mutational patterns and the relationship between indels and cancer.

Indel segregating within introns in the chicken genome are positively correlated with the recombination rates

Insertions and deletions (Indel) are important sources of genetic diversity and phenotypic divergence. Many factors such as mutation, recombination, selection and genetic drift can jointly affect the indel distribution across the genome. Studies of the relationship between recombination and indel density can, to a certain extent, reflect the selective constrain on indel. Based on the improved genetic map, genome sequence assembly and the partial (0.25X) shotgun sequencing of three breeds of domestic chicken, we calculated the recombination rates and the indel density segregating within introns and intergenic for 4 Mb windows (n = 210). Regression analyses demonstrated that recombination rates are significantly correlated with intron indel density, but not with the intergenic indel density. After adjusted regional effect, the significant trend was remained. This implies that selection is an important factor to influence the indel distribution within introns in chicken genome. By contrast, the intergenic indel seem to be neutral. Since the intron indel density on Z chromosome is less than half of that on autosomes, we preliminarily deduced that genetic hitchhiking might be more important than background selection in producing the observed correlation. As these two processes are not mutually exclusive, it is most likely that both contribute somewhat to the observed pattern. In result similar to previous study, we also found SNP density is highly correlated with indel density. Based on this characteristic, a hypothesis suggested that there are common effects of mutation and/or selection on the occurrence of indel and point mutations. This hypothesis can not explain our observations.

Indel segregating within introns in the chicken genome are positively correlated with the recombination rates

Insertions and deletions (Indel) are important sources of genetic diversity and phenotypic divergence. Many factors such as mutation, recombination, selection and genetic drift can jointly affect the indel distribution across the genome. Studies of the relationship between recombination and indel density can, to a certain extent, reflect the selective constrain on indel. Based on the improved genetic map, genome sequence assembly and the partial (0.25X) shotgun sequencing of three breeds of domestic chicken, we calculated the recombination rates and the indel density segregating within introns and intergenic for 4 Mb windows (n = 210). Regression analyses demonstrated that recombination rates are significantly correlated with intron indel density, but not with the intergenic indel density. After adjusted regional effect, the significant trend was remained. This implies that selection is an important factor to influence the indel distribution within introns in chicken genome. By contrast, the intergenic indel seem to be neutral. Since the intron indel density on Z chromosome is less than half of that on autosomes, we preliminarily deduced that genetic hitchhiking might be more important than background selection in producing the observed correlation. As these two processes are not mutually exclusive, it is most likely that both contribute somewhat to the observed pattern. In result similar to previous study, we also found SNP density is highly correlated with indel density. Based on this characteristic, a hypothesis suggested that there are common effects of mutation and/or selection on the occurrence of indel and point mutations. This hypothesis can not explain our observations.

Is morphology telling the truth about the evolution of the species rich genus Peperomia (Piperaceae)?

Peperomia is with approximately 1,600 species one of the species rich angiosperm genera. Several characters on which current infrageneric classifications are based are influenced by parallel evolution. A well-resolved molecular backbone phylogeny of the genus is needed to address evolutionary questions about morphological traits. Based on separate and combined analyses of a morphological data set and three molecular data sets, phylogenetic relationships within Peperomia are investigated with respect to character evolution. The resulting trees from different datasets are highly congruent. Morphological characters are mapped on a combined molecular tree, visualizing the contrast between previously used homoplastic characters and some newly observed characters, that can be used to delimit monophyletic groups. Length mutational events of the chloroplast dataset are coded and plotted on the respective tree, to test if indels support alternative hypothesis of relationships found in the nuclear datasets as well as the overall performance of indels compared with substitutional mutations. Our findings indicate that length distribution of indels is highest among five and six bp events. Autapomorphic and synapomorphic length mutations are most frequent in both insertions and deletions and are also more frequent independent of the length of the mutation. Concluding, independent of the length, mutations are of phylogenetic importance and should not be disregarded. None of the homoplastic indels turn into synapomorphic indels, supporting the different topology of the nrDNA tree but indicate areas of molecular evolution in favour of length mutations resulting in independent events.