Performance of Common Analysis Methods for Detecting Low-Frequency Single Nucleotide Variants in Targeted Next-Generation Sequence Data

Next-generation sequencing (NGS) is becoming a common approach for clinical testing of oncology specimens for mutations in cancer genes. Unlike inherited variants, cancer mutations may occur at low frequencies because of contamination from normal cells or tumor heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (>1000×) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with >98% sensitivity and >99% positive predictive value in coding regions. Coverage analysis demonstrated that >500× coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads before variant identification. Finally, we demonstrate the utility of high-sensitivity variant callers with data from 15 clinical lung cancers

Ultracontinuous single haplotype genome assemblies for the domestic cat (Felis catus) and Asian leopard cat (Prionailurus bengalensis)

In addition to including one of the most popular companion animals, species from the cat family Felidae serve as a powerful system for genetic analysis of inherited and infectious disease, as well as for the study of phenotypic evolution and speciation. Previous diploid-based genome assemblies for the domestic cat have served as the primary reference for genomic studies within the cat family. However, these versions suffered from poor resolution of complex and highly repetitive regions, with substantial amounts of unplaced sequence that is polymorphic or copy number variable. We sequenced the genome of a female F1 Bengal hybrid cat, the offspring of a domestic cat (Felis catus) x Asian leopard cat (Prionailurus bengalensis) cross, with PacBio long sequence reads and used Illumina sequence reads from the parents to phase \u3e99.9% of the reads into the two species’ haplotypes. De novo assembly of the phased reads produced highly continuous haploid genome assemblies for the domestic cat and Asian leopard cat, with contig N50 statistics exceeding 83 Mb for both genomes. Whole genome alignments reveal the Felis and Prionailurus genomes are colinear, and the cytogenetic differences between the homologous F1 and E4 chromosomes represent a case of centromere repositioning in the absence of a chromosomal inversion. Both assemblies offer significant improvements over the previous domestic cat reference genome, with a 100% increase in contiguity and the capture of the vast majority of chromosome arms in one or two large contigs. We further demonstrated that comparably accurate F1 haplotype phasing can be achieved with members of the same species when one or both parents of the trio are not available. These novel genome resources will empower studies of feline precision medicine, adaptation and speciation

RAG-induced DNA double-strand breaks signal through Pim2 to promote pre-B cell survival and limit proliferation

Interleukin 7 (IL-7) promotes pre–B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre–B cell survival, how cells survive during IgL chain gene rearrangement remains unclear. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre–B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre–B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre–B cell proliferation and survival

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes

RAB26 and RAB3D Are Direct Transcriptional Targets of MIST1 That Regulate Exocrine Granule Maturation▿ †

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1−/− ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment

Transcription factor MIST1 in terminal differentiation of mouse and human plasma cells

Despite their divergent developmental ancestry, plasma cells and gastric zymogenic (chief) cells share a common function: high-capacity secretion of protein. Here we show that both cell lineages share increased expression of a cassette of 269 genes, most of which regulate endoplasmic reticulum (ER) and Golgi function, and they both induce expression of the transcription factors X-box binding protein 1 (Xbp1) and Mist1 during terminal differentiation. XBP1 is known to augment plasma cell function by establishing rough ER, and MIST1 regulates secretory vesicle trafficking in zymogenic cells. We examined morphology and function of plasma cells in wild-type and Mist1−/− mice and found subtle differences in ER structure but no overall defect in plasma cell function, suggesting that Mist1 may function redundantly in plasma cells. We next reasoned that MIST1 might be useful as a novel and reliable marker of plasma cells. We found that MIST1 specifically labeled normal plasma cells in mouse and human tissues, and, moreover, its expression was also characteristic of plasma cell differentiation in a cohort of 12 human plasma cell neoplasms. Overall, our results show that MIST1 is enriched upon plasma cell differentiation as a part of a genetic program facilitating secretory cell function and also that MIST1 is a novel marker of normal and neoplastic plasma cells in mouse and human tissues

Derivation of extra-embryonic and intra-embryonic macrophage lineages from human pluripotent stem cells.

Tissue-resident macrophages are increasingly recognized as important determinants of organ homeostasis, tissue repair, remodeling and regeneration. Although the ontogeny and function of tissue-resident macrophages has been identified as distinct from postnatal hematopoiesis, the inability to specify, in vitro, similar populations that recapitulate these developmental waves has limited our ability to study their function and potential for regenerative applications. We took advantage of the concept that tissue-resident macrophages and monocyte-derived macrophages originate from distinct extra-embryonic and definitive hematopoietic lineages to devise a system to generate pure cultures of macrophages that resemble tissue-resident or monocyte-derived subsets. We demonstrate that human pluripotent stem cell-derived extra-embryonic-like and intra-embryonic-like hematopoietic progenitors differentiate into morphologically, transcriptionally and functionally distinct macrophage populations. Single-cell RNA sequencing of developing and mature cultures uncovered distinct developmental trajectories and gene expression programs of macrophages derived from extra-embryonic-like and intra-embryonic-like hematopoietic progenitors. These findings establish a resource for the generation of human tissue resident-like macrophages to study their specification and function under defined conditions and to explore their potential use in tissue engineering and regenerative medicine applications