Allelic complexity of KMT2A partial tandem duplications in acute myeloid leukemia and myelodysplastic syndromes.

KMT2A partial tandem duplication (KMT2A-PTD) is an adverse risk factor in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), a potential therapeutic target, and an attractive marker of measurable residual disease. High initial KMT2A-PTD RNA levels have been linked to poor prognosis, but mechanisms regulating KMT2A-PTD expression are not well understood. Although KMT2A-PTD has been reported to affect only a single allele, it has been theorized but not proven that genomic gains of a monoallelic KMT2A-PTD may occur, thereby potentially driving high expression and disease progression. In this study, we identified 94 patients with KMT2A-PTDs using targeted DNA next-generation sequencing (NGS) and found that 16% (15/94) had complex secondary events, including copy-neutral loss of heterozygosity and selective gain involving the KMT2A-PTD allele. High copy numbers indicating complexity were significantly enriched in AML vs MDS and correlated with higher RNA expression. Moreover, in serial samples, complexity was associated with relapse and secondary transformation. Taken together, we provide approaches to integrate quantitative and allelic assessment of KMT2A-PTDs into targeted DNA NGS and demonstrate that secondary genetic events occur in KMT2A-PTD by multiple mechanisms that may be linked to myeloid disease progression by driving increased expression from the affected allele.

A novel TP53 tandem duplication in a child with Li-Fraumeni syndrome.

Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene TP53 are the known causal genetic defect for LFS. Single-nucleotide variants (SNVs) including missense substitutions that occur in the highly conserved DNA binding domain of the protein are the most common alterations, followed by nonsense and splice site variants. Gross copy-number changes in TP53 are rare and account for <1% of all variants. Using next-generation sequencing (NGS) panels, we identified a paternally inherited germline intragenic duplication of TP53 in a child with metastatic osteosarcoma who later developed acute myeloid leukemia (AML). Transcriptome sequencing (RNA-seq) demonstrated the duplication was tandem, encompassing exons 2-6 and 28 nt of the untranslated region (UTR) upstream of the start codon in exon 2. The inclusion of the 28 nt is expected to result in a frameshift with a stop codon 18 codons downstream from the exon 6, leading to a loss-of-function allele. This case highlights the significance of simultaneous identification of both significant copy-number variants as well as SNVs/indels using NGS panels.

Genome-Wide Identification and Characterization of the RCI2 Gene Family in Allotetraploid Brassica napus Compared with Its Diploid Progenitors.

Brassica napus and its diploid progenitors (B. rapa and B. oleracea) are suitable for studying the problems associated with polyploidization. As an important anti-stress protein, RCI2 proteins widely exist in various tissues of plants, and are crucial to plant growth, development, and stress response. In this study, the RCI2 gene family was comprehensively identified and analyzed, and 9, 9, and 24 RCI2 genes were identified in B. rapa, B. oleracea, and B. napus, respectively. Phylogenetic analysis showed that all of the identified RCI2 genes were divided into two groups, and further divided into three subgroups. Ka/Ks analysis showed that most of the identified RCI2 genes underwent a purifying selection after the duplication events. Moreover, gene structure analysis showed that the structure of RCI2 genes is largely conserved during polyploidization. The promoters of the RCI2 genes in B. napus contained more cis-acting elements, which were mainly involved in plant development and growth, plant hormone response, and stress responses. Thus, B. napus might have potential advantages in some biological aspects. In addition, the changes of RCI2 genes during polyploidization were also discussed from the aspects of gene number, gene structure, gene relative location, and gene expression, which can provide reference for future polyploidization analysis.

Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L.

BACKGROUNDS: Populus and Salix belong to Salicaceae and are used as models to investigate woody plant physiology. The variation of karyotype and nuclear DNA content can partly reflect the evolutionary history of the whole genome, and can provide critical information for understanding, predicting, and potentially ameliorating the woody plant traits. Therefore, it is essential to study the chromosome number (CN) and genome size in detail to provide information for revealing the evolutionary process of Salicaceae. RESULTS: In this study, we report the somatic CNs of seventeen species from eight genera in Salicaceae. Of these, CNs for twelve species and for five genera are reported for the first time. Among the three subfamilies of Salicaceae, the available data indicate CN in Samydoideae is n = 21, 22, 42. The only two genera, Dianyuea and Scyphostegia, in Scyphostegioideae respectively have n = 9 and 18. In Salicoideae, Populus, Salix and five genera closely related to them (Bennettiodendron, Idesia, Carrierea, Poliothyrsis, Itoa) are based on relatively high CNs from n = 19, 20, 21, 22 to n = 95 in Salix. However, the other genera of Salicoideae are mainly based on relatively low CNs of n = 9, 10, 11. The genome sizes of 35 taxa belonging to 14 genera of Salicaceae were estimated. Of these, the genome sizes of 12 genera and all taxa except Populus euphratica are first reported. Except for Dianyuea, Idesia and Bennettiodendron, all examined species have relatively small genome sizes of less than 1 pg, although polyploidization exists. CONCLUSIONS: The variation of CN and genome size across Salicaceae indicates frequent ploidy changes and a widespread sharing of the salicoid whole genome duplication (WGD) by the relatives of Populus and Salix. The shrinkage of genome size after WGD indicates massive loss of genomic components. The phylogenetic asymmetry in clade of Populus, Salix, and their close relatives suggests that there is a lag-time for the subsequent radiations after the salicoid WGD event. Our results provide useful data for studying the evolutionary events of Salicaceae.

Genome-Wide Identification and Characterization of the Cystatin Gene Family in Bread Wheat (Triticum aestivum L.).

Cystatins, as reversible inhibitors of papain-like and legumain proteases, have been identified in several plant species. Although the cystatin family plays crucial roles in plant development and defense responses to various stresses, this family in wheat (Triticum aestivum L.) is still poorly understood. In this study, 55 wheat cystatins (TaCystatins) were identified. All TaCystatins were divided into three groups and both the conserved gene structures and peptide motifs were relatively conserved within each group. Homoeolog analysis suggested that both homoeolog retention percentage and gene duplications contributed to the abundance of the TaCystatin family. Analysis of duplication events confirmed that segmental duplications played an important role in the duplication patterns. The results of codon usage pattern analysis showed that TaCystatins had evident codon usage bias, which was mainly affected by mutation pressure. TaCystatins may be regulated by cis-acting elements, especially abscisic acid and methyl jasmonate responsive elements. In addition, the expression of all selected TaCystatins was significantly changed following viral infection and cold stress, suggesting potential roles in response to biotic and abiotic challenges. Overall, our work provides new insights into TaCystatins during wheat evolution and will help further research to decipher the roles of TaCystatins under diverse stress conditions.

Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing.

Genomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

Fms-like tyrosine kinase 3-internal tandem duplications epigenetically activates checkpoint kinase 1 in acute myeloid leukemia cells.

It is not clear how Fms-like tyrosine kinase 3-internal tandem duplications (FLT3-ITD) regulates checkpoint kinase 1 (CHK1) in acute myeloid leukemia (AML). In this study, we investigated the regulatory effect of FLT3-ITD on CHK1. Our results showed that CHK1 was highly expressed in FLT3-ITD positive AML. The overall survival rate and disease-free survival rate of AML patients with high CHK1 level were lower than those of patients with low CHK1 level. Mechanistically, FLT3-ITD recruited p300 to the CHK1 promoter and subsequently acetylated H3K27, thereby enhancing the transcription of CHK1. Interfering with the expression of CHK1 significantly inhibited the cell proliferation and induced cell apoptosis in FLT3-ITD positive MV4-11 cells. In addition, CHK1 knockdown promoted the sensitivity of MV4-11 cells to the epigenetic inhibitors JQ1 and C646. This study discovers a new therapeutic target for FLT3-ITD + AML and provided evidence for the combination of epigenetic inhibitors for AML treatment.

All-trans Retinoic Acid, Arsenic Trioxide, and Anthracycline-based Chemotherapy Improves Outcome in Newly Diagnosed Acute Promyelocytic Leukemia Regardless of FLT3-ITD Mutation Status.

All-trans retinoic acid (ATRA) and pre-upfront arsenic trioxide (ATO) have revolutionized the therapy of acute promyelocytic leukemia (APL). However, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutations is associated with increased risk of relapse. The aim of this study was to analyze the prognostic impact of FLT3-ITD on APL patients who received remission induction with ATRA, idarubicin (IDA) and/or ATO, followed by ATRA plus ATO along with anthracycline, as consolidation therapy. A total of 72 patients newly diagnosed with APL were included in this study. 83.3% of the patients achieved complete remission (CR) after induction therapy. FLT3-ITD mutations were detected in 16 (22.2%) patients and closely related to bcr-3 PML-RARa transcript (P<0.001). The 5-year overall survival (OS) rate was 100% in both FLT3-ITDpositive and FLT3-ITDnegative groups, and there was no significant difference in 5-year event-free survival (EFS) between the two groups (78.3% vs. 83.3%, P=0.85). ATRA plus ATO and anthracycline-based chemotherapy achieved great outcome in newly diagnosed APL regardless of the FLT3-ITD mutation status.

A novel BRCA1 duplication and new insights on the spectrum and frequency of germline large genomic rearrangements in BRCA1/BRCA2.

Heritable breast cancers account for 5% to 10% of all breast cancers, and monogenic, highly penetrant genes cause them. Around 90% of pathogenic variants in BRCA1 and BRCA2 are observed using gene sequencing, with another 10% identified through gene duplication/deletion analysis, which differs across various communities. In this study, we performed a next-generation sequencing panel and MLPA on 1484 patients to explain the importance of recurrent germline duplications/deletions of BRCA1-2 and their clinical results and determine how often BRCA gene LGRs were seen in people suspected of hereditary breast and ovarian cancer syndrome. The large genomic rearrangements (LGRs) frequency was approximately 1% (14/1484). All 14 mutations were heterozygous and detected in patients with breast cancer. BRCA1 mutations were more predominant (n = 8, 57.1%) than BRCA2 mutations (6, 42.9%). The most common recurrent mutations were BRCA2 exon three and BRCA1 exon 24 (23) deletions. To the best of our knowledge, BRCA1 5'UTR-exon11 duplication has never been reported before. Testing with MLPA is essential to identify patients at high risk. Our data demonstrate that BRCA1-2 LGRs should be considered when ordering genetic testing for individuals with a personal or family history of cancer, particularly breast cancer. Further research could shed light on BRCA1-2 LGRs' unique carcinogenesis roles.

BCOR Internal Tandem Duplication Expression in Neural Stem Cells Promotes Growth, Invasion, and Expression of PRC2 Targets.

Central nervous system tumor with BCL6-corepressor internal tandem duplication (CNS-BCOR ITD) is a malignant entity characterized by recurrent alterations in exon 15 encoding the essential binding domain for the polycomb repressive complex (PRC). In contrast to deletion or truncating mutations seen in other tumors, BCOR expression is upregulated in CNS-BCOR ITD, and a distinct oncogenic mechanism has been suggested. However, the effects of this change on the biology of neuroepithelial cells is poorly understood. In this study, we introduced either wildtype BCOR or BCOR-ITD into human and murine neural stem cells and analyzed them with quantitative RT-PCR and RNA-sequencing, as well as growth, clonogenicity, and invasion assays. In human cells, BCOR-ITD promoted derepression of PRC2-target genes compared to wildtype BCOR. A similar effect was found in clinical specimens from previous studies. However, no growth advantage was seen in the human neural stem cells expressing BCOR-ITD, and long-term models could not be established. In the murine cells, both wildtype BCOR and BCOR-ITD overexpression affected cellular differentiation and histone methylation, but only BCOR-ITD increased cellular growth, invasion, and migration. BCOR-ITD overexpression drives transcriptional changes, possibly due to altered PRC function, and contributes to the oncogenic transformation of neural precursors.

Confined placental mosaicism involving multiple de novo copy number variants associated with fetal growth restriction: A case report.

The presence of multiple large (>1 Mb) copy number variants (CNVs) in non-malignant tissue is rare in human genetics. We present a liveborn male with a birth weight below the first percentile associated with placental mosaicism involving eight 2.4-3.9 Mb de novo duplications. We found that the duplications likely co-localized to the same cells, were mosaic in the placenta, and impacted maternal and paternal chromosomes. In addition, 27.4 Mb and 240 genes were duplicated in affected cells, including candidate placental genes KISS1 and REN. We ruled out involvement of homologous recombination-based mechanisms or an altered epigenome in generating the CNVs. This case highlights the diversity of genetic abnormalities in the human placenta and the gaps in our knowledge of how such errors arise.

RAD21 is a driver of chromosome 8 gain in Ewing sarcoma to mitigate replication stress.

Aneuploidy, defined as whole-chromosome gain or loss, causes cellular stress but, paradoxically, is a frequent occurrence in cancers. Here, we investigate why ∼50% of Ewing sarcomas, driven by the EWS-FLI1 fusion oncogene, harbor chromosome 8 gains. Expression of the EWS-FLI1 fusion in primary cells causes replication stress that can result in cellular senescence. Using an evolution approach, we show that trisomy 8 mitigates EWS-FLI1-induced replication stress through gain of a copy of RAD21. Low-level ectopic expression of RAD21 is sufficient to dampen replication stress and improve proliferation in EWS-FLI1-expressing cells. Conversely, deleting one copy in trisomy 8 cells largely neutralizes the fitness benefit of chromosome 8 gain and reduces tumorgenicity of a Ewing sarcoma cancer cell line in soft agar assays. We propose that RAD21 promotes tumorigenesis through single gene copy gain. Such genes may explain some recurrent aneuploidies in cancer.

Altered chromatin conformation and transcriptional regulation in watermelon following genome doubling.

Polyploidy has played a crucial role in plant evolution, development and function. Synthetic autopolyploid represents an ideal system to investigate the effects of polyploidization on transcriptional regulation. In this study, we deciphered the impact of genome duplication at phenotypic and molecular levels in watermelon. Overall, 88% of the genes in tetraploid watermelon followed a >1:1 dosage effect, and accordingly, differentially expressed genes were largely upregulated. In addition, a great number of hypomethylated regions (1688) were identified in an isogenic tetraploid watermelon. These differentially methylated regions were localized in promoters and intergenic regions and near transcriptional start sites of the identified upregulated genes, which enhances the importance of methylation in gene regulation. These changes were reflected in sophisticated higher-order chromatin structures. The genome doubling caused switching of 108 A and 626 B compartments that harbored genes associated with growth, development and stress responses.

Genome-wide identification and characterization of olfactory receptor genes in common carp (Cyprinus carpio).

The environment contains a large extent of chemical information, which could be detected as olfactory sense. Olfactory in vertebrates plays important roles on many aspects during life time, including localizing prey or food, avoiding predators, mating behavior and social communication. Considering the essential role of olfactory receptors in the specific recognition of diverse stimuli, understanding the evolutionary dynamics of olfactory receptors in teleost means a lot, especially in the allotetraploid common carp, who has undergone the fourth whole-genome duplication event. Here, we identified the whole set of olfactory receptor genes in representative teleosts and found a significant contraction in common carp when compared with other teleosts. Odorant receptor genes (OR) occupy the most among four groups of olfactory receptors, including 33 functional genes and 16 pseudogenes. Furthermore, 6 trace amine-associated receptor (TAAR) genes (including 1 pseudogene), 7 odorant-related-A receptor genes, and 10 olfactory C family receptor genes (including 3 pseudogenes) were identified in common carp. Phylogenetic and motif analysis were performed to illustrate the phylogenetic relationship and structural conservation of teleost olfactory receptors. Selection pressure analysis suggested that olfactory receptor groups in common carp were all under relaxed purifying-selection. Additionally, gene expression divergences for olfactory receptor genes were investigated during embryonic development stages of common carp. We aim to determine the abundance of common carp olfactory receptor genes, explore the evolutionary fate and expression dynamics, and provide some genomic clues for the evolution of polyploid olfactory after whole-genome duplication and for future studies of teleost olfactory.

Phylotranscriptomic insights into Asteraceae diversity, polyploidy, and morphological innovation.

Biodiversity is not evenly distributed among related groups, raising questions about the factors contributing to such disparities. The sunflower family (Asteraceae, >26,000 species) is among the largest and most diverse plant families, but its species diversity is concentrated in a few subfamilies, providing an opportunity to study the factors affecting biodiversity. Phylotranscriptomic analyses here of 244 transcriptomes and genomes produced a phylogeny with strong support for the monophyly of Asteraceae and the monophyly of most subfamilies and tribes. This phylogeny provides a reference for detecting changes in diversification rates and possible factors affecting Asteraceae diversity, which include global climate shifts, whole-genome duplications (WGDs), and morphological evolution. The origin of Asteraceae was estimated at ~83 Mya, with most subfamilies having diverged before the Cretaceous-Paleocene boundary. Phylotranscriptomic analyses supported the existence of 41 WGDs in Asteraceae. Changes to herbaceousness and capitulescence with multiple flower-like capitula, often with distinct florets and scaly pappus/receptacular bracts, are associated with multiple upshifts in diversification rate. WGDs might have contributed to the survival of early Asteraceae by providing new genetic materials to support morphological transitions. The resulting competitive advantage for adapting to different niches would have increased biodiversity in Asteraceae.

Horseshoe crab genomes reveal the evolution of genes and microRNAs after three rounds of whole genome duplication.

Whole genome duplication (WGD) has occurred in relatively few sexually reproducing invertebrates. Consequently, the WGD that occurred in the common ancestor of horseshoe crabs ~135 million years ago provides a rare opportunity to decipher the evolutionary consequences of a duplicated invertebrate genome. Here, we present a high-quality genome assembly for the mangrove horseshoe crab Carcinoscorpius rotundicauda (1.7 Gb, N50 = 90.2 Mb, with 89.8% sequences anchored to 16 pseudomolecules, 2n = 32), and a resequenced genome of the tri-spine horseshoe crab Tachypleus tridentatus (1.7 Gb, N50 = 109.7 Mb). Analyses of gene families, microRNAs, and synteny show that horseshoe crabs have undergone three rounds (3R) of WGD. Comparison of C. rotundicauda and T. tridentatus genomes from populations from several geographic locations further elucidates the diverse fates of both coding and noncoding genes. Together, the present study represents a cornerstone for improving our understanding of invertebrate WGD events on the evolutionary fates of genes and microRNAs, at both the individual and population level. We also provide improved genomic resources for horseshoe crabs, of applied value for breeding programs and conservation of this fascinating and unusual invertebrate lineage.

Proteus syndrome caused by novel somatic AKT1 duplication.

Proteus syndrome (PS) is a rare overgrowth disorder that presents with asymmetrical growth of the bone and fat tissues following a mosaic pattern mutation. The estimated worldwide incidence is approximately one in one million live births. Proteus syndrome causes disfigurement and psychological impact through its effects on somatic tissue. Due to its rarity and diversity of tissues involved, it represents a significant challenge to caregivers and multidisciplinary medical teams. Here, we report a Saudi girl, with a large left cervical mass discovered antenatally. This mass was identified as a growing cystic hygroma, and she had features of overgrowth and hemangiomas. Whole exome sequencing was negative from the blood lymphocytes and affected tissue sample.  However, deletion duplication analysis from tissue shows a novel mosaic somatic mutation of the AKT1 gene. Somatic mutation remains an obstacle, and the geneticist has an essential role in its management, providing an established genetic diagnosis, prognosis, and family counselling.

Apple Autotetraploids with Enhanced Resistance to Apple Scab (Venturia inaequalis) Due to Genome Duplication-Phenotypic and Genetic Evaluation.

Among the fungal diseases of apple trees, serious yield losses are due to an apple scab caused by Venturia inaequalis. Protection against this disease is based mainly on chemical treatments, which are currently very limited. Therefore, it is extremely important to introduce cultivars with reduced susceptibility to this pathogen. One of the important sources of variability for breeding is the process of polyploidization. Newly obtained polyploids may acquire new features, including increased resistance to diseases. In our earlier studies, numerous tetraploids have been obtained for several apple cultivars with 'Free Redstar' tetraploids manifesting enhanced resistance to apple scab. In the present study, tetraploids of 'Free Redstar' were assessed in terms of phenotype and genotype with particular emphasis on the genetic background of their increased resistance to apple scab. Compared to diploid plants, tetraploids (own-rooted plants) were characterized with poor growth, especially during first growing season. They had considerably shorter shoots, fewer branches, smaller stem diameter, and reshaped leaves. In contrast to own-rooted plants, in M9-grafted three-year old trees, no significant differences between diplo- and tetraploids were observed, either in morphological or physiological parameters, with the exceptions of the increased leaf thickness and chlorophyll content recorded in tetraploids. Significant differences between sibling tetraploid clones were recorded, particularly in leaf shape and some physiological parameters. The amplified fragment length polymorphism (AFLP) analysis confirmed genetic polymorphism of tetraploid clones. Methylation-sensitive amplification polymorphism (MSAP) analysis showed that the level of DNA methylation was twice as high in young tetraploid plants as in a diploid donor tree, which may explain the weaker vigour of neotetraploids in the early period of their growth in the juvenile phase. Molecular analysis showed that 'Free Redstar' cultivar and their tetraploids bear six Rvi genes (Rvi5, Rvi6, Rvi8, Rvi11, Rvi14 and Rvi17). Transcriptome analysis confirmed enhanced resistance to apple scab of 'Free Redstar' tetraploids since the expression levels of genes related to resistance were strongly enhanced in tetraploids compared to their diploid counterparts.

The fragility of a structurally diverse duplication block triggers recurrent genomic amplification.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.

New Approaches for Inferring Phylogenies in the Presence of Paralogs.

The availability of whole genome sequences was expected to supply essentially unlimited data for phylogenetics. However, strict reliance on single-copy genes for this purpose has drastically limited the amount of data that can be used. Here, we review several approaches for increasing the amount of data used for phylogenetic inference, focusing on methods that allow for the inclusion of duplicated genes (paralogs). Recently developed methods that are robust to high levels of incomplete lineage sorting also appear to be robust to the inclusion of paralogs, suggesting a promising way to take full advantage of genomic data. We discuss the pitfalls of these approaches, as well as further avenues for research.