High-resolution profiling and discovery of planarian small RNAs

Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are down-regulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.     

Exome and genome sequencing: a revolution for the discovery and diagnosis of monogenic disorders

Massively parallel DNA sequencing has revolutionized analyses of human genetic variation. From having been out of reach for individual research groups and even more so for clinical diagnostic laboratories until recently, it is now possible to analyse complete human genomes within reasonable time frames and at a reasonable cost using technologies that are becoming increasingly available. This represents a huge advance in our ability to provide correct diagnoses for patients with rare inherited disorders and their families. This paradigm shift is especially dramatic within the area of monogenic disorders. Not only can rapid and safe diagnostics of virtually all known single‐gene defects now be established, but novel causes of disease in previously unsolved cases can also be identified, illuminating novel pathways important for normal physiology. This greatly increases the capability not only to improve management of rare disorders, but also to improve understanding of pathogenetic mechanisms relevant for common, complex diseases.     

Plasma DNA aberrations in systemic lupus erythematosus revealed by genomic and methylomic sequencing

We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double–stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non–IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non–IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients.Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that has the potential of affecting multiple organ systems, including the skin, muscles, bones, lungs, kidneys, as well as the cardiovascular and central nervous systems (1, 2). SLE can cause various tissue inflammation and damages in a chronic manner. Renal complications, infections, myocardial infarctions, and central nervous system involvement are the major causes of death in SLE patients (3). The extremely variable clinical manifestations and the absence of effective tests to monitor disease activity present a challenge for clinical management (2, 3).The etiology of SLE remains unknown and is multifactorial, involving genetic, epigenetic, environmental, hormonal, and immunologic factors (2, 4). Cell death has been regarded as an important event in the pathogenesis of SLE, as it leads to the release of antigens, such as nucleic acids, for immune complex formation, which may trigger a cascade of immune responses against the bodily tissues of the SLE patients (5, 6). Defects in the mechanism of cell death (7), impairment in the clearance of dead cells (8), and deficiency in DNase activity (9) have been implicated in SLE and suggested to be involved in the generation of autoantigens (5, 6).In addition, epigenetic regulation is an important mechanism for maintaining the normal functioning of the immune system. Perturbation of the epigenetic regulation can disrupt the immunologic self-tolerance (10). Following the demonstration of impaired DNA methylation of T cells in SLE patients (11), an increasing amount of evidence has highlighted the contribution of epigenetic mechanisms in this disorder (12, 13). Hypomethylated apoptotic DNA from cells has been shown to be potentially pathogenic and may provoke the humoral and cellular immune responses in SLE (14).SLE was one of the pathological conditions reported to be associated with the presence of circulating DNA nearly 50 years ago (15). Since then, studies using various detection methods have demonstrated the elevations of circulating DNA in SLE patients (16, 17). In addition, early reports have highlighted that the circulating DNA that form immune complexes with autoantibodies in SLE patients displays a characteristic fragmentation pattern that resembles the DNA laddering pattern of apoptosis by gel electrophoresis (18). These findings have suggested an interplay of apoptosis and circulating DNA in the pathogenesis of SLE. However, there have been very few studies reporting the detailed biological characterization of circulating DNA in SLE.The advent of massively parallel sequencing has enabled the investigation of circulating DNA at single-base resolution on a genome-wide scale in fields such as noninvasive prenatal testing (19–21) and cancer detection (22–25). It would be of great interest to use this technology to explore the genomic and methylomic features of plasma DNA in SLE patients. In particular, the interplay of deregulated cell death, altered epigenetic regulation and production of autoimmune antibodies in SLE patients might cause abnormal patterns of circulating DNA. Hence, in this study we delineated the biological characteristics of DNA in the plasma of SLE patients using genome-wide genomic and methylomic sequencing.     

Patient‐tailored analysis of minimal residual disease in acute myeloid leukemia using next‐generation sequencing

Next‐generation sequencing techniques have revealed that leukemic cells in acute myeloid leukemia often are characterized by a limited number of somatic mutations. These mutations can be the basis for the detection of leukemic cells in follow‐up samples. The aim of this study was to identify leukemia‐specific mutations in cells from patients with acute myeloid leukemia and to use these mutations as markers for minimal residual disease. Leukemic cells and normal lymphocytes were simultaneously isolated at diagnosis from 17 patients with acute myeloid leukemia using fluorescence‐activated cell sorting. Exome sequencing of these cells identified 240 leukemia‐specific single nucleotide variations and 22 small insertions and deletions. Based on estimated allele frequencies and their accuracies, 191 of these mutations qualified as candidates for minimal residual disease analysis. Targeted deep sequencing with a significance threshold of 0.027% for single nucleotide variations and 0.006% for NPM1 type A mutation was developed for quantification of minimal residual disease. When tested on follow‐up samples from a patient with acute myeloid leukemia, targeted deep sequencing of single nucleotide variations as well as NPM1 was more sensitive than minimal residual disease quantification with multiparameter flow cytometry. In conclusion, we here describe how exome sequencing can be used for identification of leukemia‐specific mutations in samples already at diagnosis of acute myeloid leukemia. We also show that targeted deep sequencing of such mutations, including single nucleotide variations, can be used for high‐sensitivity quantification of minimal residual disease in a patient‐tailored manner.     

低深度全基因组测序检测以甲状腺功能减退起病的Williams-Beuren综合征一例

目的探讨1例不明原因智力低下、生长发育迟缓、具有特殊面容及甲状腺功能减退的5岁男性患儿的临床表型和遗传学病因。方法用常规G显带技术分析患儿及其父母染色体核型,应用全外显子组测序及低深度全基因组测序技术(low-coverage massively parallel CNV sequencing,CNV-seq)对患儿进行可能致病突变及染色体拷贝数变异的分析。结果患儿染色体核型为46,XY,其父母未见明显的染色体异常。CNV-seq分析显示患儿在染色体7q11.23区域存在大小为1.56 Mb的杂合性缺失,缺失区域包含24个编码蛋白质的基因,其缺失与Williams-Beuren综合征相关。通过对其父母检测CNV-seq,发现该缺失为一新发缺失。结论本研究用CNV-seq技术确诊了1例以甲状腺功能减退起病的Williams-Beuren综合征患儿,有助于提高临床医师对Williams-Beuren综合征表型与发病机制的认识。     

Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome

The genome of soybean (Glycine max), a commercially important crop, has recently been sequenced and is one of six crop species to have been sequenced. Here we report the genome sequence of G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932). The 48.8-Gb Illumina Genome Analyzer (Illumina-GA) short DNA reads were aligned to the G. max reference genome and a consensus was determined for G. soja. This consensus sequence spanned 915.4 Mb, representing a coverage of 97.65% of the G. max published genome sequence and an average mapping depth of 43-fold. The nucleotide sequence of the G. soja genome, which contains 2.5 Mb of substituted bases and 406 kb of small insertions/deletions relative to G. max, is ～0.31% different from that of G. max. In addition to the mapped 915.4-Mb consensus sequence, 32.4 Mb of large deletions and 8.3 Mb of novel sequence contigs in the G. soja genome were also detected. Nucleotide variants of G. soja versus G. max confirmed by Roche Genome Sequencer FLX sequencing showed a 99.99% concordance in single-nucleotide polymorphism and a 98.82% agreement in insertion/deletion calls on Illumina-GA reads. Data presented in this study suggest that the G. soja/G. max complex may be at least 0.27 million y old, appearing before the relatively recent event of domestication (6,000～9,000 y ago). This suggests that soybean domestication is complicated and that more in-depth study of population genetics is needed. In any case, genome comparison of domesticated and undomesticated forms of soybean can facilitate its improvement.     

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm

A common challenge in pathogen discovery by deep sequencing approaches is to recognize viral or subviral pathogens in samples of diseased tissue that share no significant homology with a known pathogen. Here we report a homology-independent approach for discovering viroids, a distinct class of free circular RNA subviral pathogens that encode no protein and are known to infect plants only. Our approach involves analyzing the sequences of the total small RNAs of the infected plants obtained by deep sequencing with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Viroid infection triggers production of viroid-derived overlapping siRNAs that cover the entire genome with high densities. PFOR retains viroid-specific siRNAs for genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, which is synthesized from circular or multimeric repeated-sequence templates during viroid replication. We show that viroids from the two known families are readily identified and their full-length sequences assembled by PFOR from small RNAs sequenced from infected plants. PFOR analysis of a grapevine library further identified a viroid-like circular RNA 375 nt long that shared no significant sequence homology with known molecules and encoded active hammerhead ribozymes in RNAs of both plus and minus polarities, which presumably self-cleave to release monomer from multimeric replicative intermediates. A potential application of the homology-independent approach for viroid discovery in plant and animal species where RNA replication triggers the biogenesis of siRNAs is discussed.     

Breast cancer signatures for invasiveness and prognosis defined by deep sequencing of microRNA

The transition from ductal carcinoma in situ to invasive ductal carcinoma is a key event in breast cancer progression that is still not well understood. To discover the microRNAs regulating this critical transition, we used 80 biopsies from invasive ductal carcinoma, 8 from ductal carcinoma in situ, and 6 from normal breast. We selected them from a recently published deep-sequencing dataset [Farazi TA, et al. (2011) Cancer Res 71:4443-4453]. The microRNA profile established for the normal breast to ductal carcinoma in situ transition was largely maintained in the in situ to invasive ductal carcinoma transition. Nevertheless, a nine-microRNA signature was identified that differentiated invasive from in situ carcinoma. Specifically, let-7d, miR-210, and -221 were down-regulated in the in situ and up-regulated in the invasive transition, thus featuring an expression reversal along the cancer progression path. Additionally, we identified microRNAs for overall survival and time to metastasis. Five noncoding genes were associated with both prognostic signatures--miR-210, -21, -106b*, -197, and let-7i, with miR-210 the only one also involved in the invasive transition. To pinpoint critical cellular functions affected in the invasive transition, we identified the protein coding genes with inversely related profiles to miR-210: BRCA1, FANCD, FANCF, PARP1, E-cadherin, and Rb1 were all activated in the in situ and down-regulated in the invasive carcinoma. Additionally, we detected differential splicing isoforms with special features, including a truncated EGFR lacking the kinase domain and overexpressed only in ductal carcinoma in situ.     

Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients

The analysis of tumor-derived circulating cell-free DNA opens up new possibilities for performing liquid biopsies for the assessment of solid tumors. Although its clinical potential has been increasingly recognized, many aspects of the biological characteristics of tumor-derived cell-free DNA remain unclear. With respect to the size profile of such plasma DNA molecules, a number of studies reported the finding of increased integrity of tumor-derived plasma DNA, whereas others found evidence to suggest that plasma DNA molecules released by tumors might be shorter. Here, we performed a detailed analysis of the size profiles of plasma DNA in 90 patients with hepatocellular carcinoma, 67 with chronic hepatitis B, 36 with hepatitis B-associated cirrhosis, and 32 healthy controls. We used massively parallel sequencing to achieve plasma DNA size measurement at single-base resolution and in a genome-wide manner. Tumor-derived plasma DNA molecules were further identified with the use of chromosome arm-level z-score analysis (CAZA), which facilitated the studying of their specific size profiles. We showed that populations of aberrantly short and long DNA molecules existed in the plasma of patients with hepatocellular carcinoma. The short ones preferentially carried the tumor-associated copy number aberrations. We further showed that there were elevated amounts of plasma mitochondrial DNA in the plasma of hepatocellular carcinoma patients. Such molecules were much shorter than the nuclear DNA in plasma. These results have improved our understanding of the size profile of tumor-derived circulating cell-free DNA and might further enhance our ability to use plasma DNA as a molecular diagnostic tool.Analysis of circulating cell-free DNA has been increasingly used for the detection and monitoring of cancers (1–5). Different cancer-associated molecular characteristics, including copy number aberrations (6–9), methylation changes (10–13), single-nucleotide mutations (6, 14–17), cancer-derived viral sequences (18, 19), and chromosomal rearrangements (20, 21), can be detected in the plasma of patients with various types of cancers. Despite the rapid expansion of clinical applications, many fundamental molecular characteristics of circulating DNA in cancer patients remain unclear. In particular, previous studies on the size of circulating DNA in cancer patients gave inconsistent results. Studies have demonstrated that the overall integrity of circulating DNA would increase in cancer patients compared with subjects without a malignant condition (22–25). Using PCR with different amplicon sizes, it was shown that the proportion of longer DNA would be higher in cancer patients. This aberration in DNA integrity was shown to be reversible after treatment, and the persistence of such changes was associated with poor prognosis (22, 26). On the other hand, there is also seemingly contradictory evidence that circulating DNA derived from tumor tissues might be shorter than those derived from nonmalignant cells. For example, it has been shown that the proportion of DNA molecules carrying cancer-associated mutations would be higher when those mutations were detected using PCR with shorter amplicons (14, 27).In this study, we aimed to reconcile these apparent inconsistencies through the use of a study design that takes advantage of the following: (i) genome-wide high-resolution size profiling of plasma DNA enabled by massively parallel sequencing (28, 29); and (ii) an efficient approach to distinguish tumor-derived DNA from the nontumoral background DNA in the plasma of cancer patients. We believe that enhanced characterization of plasma DNA molecules in cancer patients would be useful for understanding the mechanisms involved in their generation and would offer useful insights for the development of diagnostic approaches.It has become feasible to measure the lengths of every individual plasma DNA molecule in samples with the use of massively parallel sequencing (28, 29). Hence, plasma DNA sizes could be studied in a genome-wide manner and at single-base resolution. Using this approach, the size of circulating DNA has generally been shown to resemble the size of mononucleosomal DNA, suggesting that plasma DNA might be generated through apoptosis (28, 29). In pregnant women, plasma DNA derived from the fetus has been shown to be shorter than that of DNA derived from the mother (28). The size difference between circulating fetal and maternal DNA has provided a previously unidentified conceptual basis for quantifying fetal DNA in maternal plasma and detecting chromosomal aneuploidies through size analysis of plasma DNA (30). In addition, differences in the size distributions of circulating DNA derived from the transplanted organs and the patients’ own tissues have been observed for recipients of solid organ or bone marrow transplantation (29).In this study, we used hepatocellular carcinoma (HCC) as a model to study the size distribution of plasma DNA in cancer patients. The size distributions of plasma DNA in HCC patients, patients with chronic hepatitis B virus (HBV) infection, patients with liver cirrhosis, and healthy subjects were also analyzed. Plasma of cancer patients contains a mixture of tumor-derived and non–tumor-derived DNA. We were particularly interested in studying the size profile of tumor-derived DNA in the plasma of the HCC patients. However, this is a challenging endeavor because tumor-derived plasma DNA could not be readily distinguished from the non–tumor-derived background DNA in plasma. The detection of cancer-specific mutations offers a genotypic means to distinguish the tumoral from the nontumoral plasma DNA. However, there are relatively few cancer-specific mutations across the genome (31–34) for the purpose of generating a broad, detailed, and yet cost-effective view of the size distribution of tumor-derived DNA.To circumvent this issue, we used chromosome arms that are affected by copy number aberrations (CNAs) to infer the difference in size distributions of tumor-derived and non–tumor-derived plasma DNA. The principle of this method is illustrated in Fig. 1. For chromosome arms that are amplified in the tumor tissues, the proportional contribution from tumor-derived DNA to plasma DNA would increase, whereas for chromosome arms that are deleted in the tumor, the contribution would decrease. Therefore, the comparison of size profiles of chromosome arms that are amplified and deleted would reflect the size difference between tumor-derived and non–tumor-derived DNA in plasma. CNA involving a whole chromosome arm or a large trunk of a chromosome arm is relatively common (35). Deletion of chromosomes 1p and 8p and amplification of chromosomes 1q and 8q are commonly observed in the HCC tissues (36–38). Thus, in this study, we focused on chromosomes 1 and 8 for the CNA and size-profiling analyses of plasma DNA. As the characteristic size profile of plasma nuclear DNA is likely to be related to histone packing, we hypothesized that the lack of histones packing for mitochondrial DNA might affect its abundance and size profile in plasma. Thus, we have also studied the size and fractional concentration of plasma mitochondrial DNA in the same cohort of subjects.Open in a separate windowFig. 1.Schematic illustration of the principle of plasma DNA size analysis in cancer patients. In cancer patients, plasma DNA is derived from both tumor (red molecules) and nontumor cells (blue molecules). Genomic regions that are amplified in the tumor tissue would contribute more tumoral DNA to plasma. Genomic regions that are deleted in the tumor tissue would contribute less DNA to plasma. Chromosome arm-level z-score analysis (CAZA) was used to determine if a chromosome arm is overrepresented or underrepresented in plasma DNA, suggestive of the presence of amplification or deletion, respectively, of the chromosome arm in the tumor. The size profiles of plasma DNA molecules originating from chromosome arms that are underrepresented (enriched for nontumor DNA) and overrepresented (enriched for tumor-derived DNA) were compared.     

Targeted deep sequencing of the PEAR1 locus for platelet aggregation in European and African American families

Coronary artery disease (CAD) remains a major cause of mortality and morbidity worldwide. The aggregation of activated platelets on a ruptured atherosclerotic plaque is a critical step in most acute cardiovascular events like myocardial infarction. Platelet aggregation both at baseline and after aspirin is highly heritable. Genome-wide association studies (GWAS) have identified a common variant within the first intron of the platelet endothelial aggregation receptor1 (PEAR1), to be robustly associated with platelet aggregation. In this study, we used targeted deep sequencing to fine-map the prior GWAS peak and identify additional rare variants of PEAR1 that account for missing heritability in platelet aggregation within the GeneSTAR families.

In this study, 1709 subjects (1043 European Americans, EA and 666 African Americans, AA) from families in the GeneSTAR study were included. In vitro platelet aggregation in response to collagen, ADP and epinephrine was measured at baseline and 14 days after aspirin therapy (81 mg/day). Targeted deep sequencing of PEAR1 in addition to 2kb of upstream and downstream of the gene was performed. Under an additive genetic model, the association of single variants of PEAR1 with platelet aggregation phenotypes were examined. Additionally, we examined the association between the burden of PEAR1 rare non-synonymous variants and platelet aggregation phenotypes.

Of 532 variants identified through sequencing, the intron 1 variant, rs12041331, was significantly associated with all platelet aggregation phenotypes at baseline and after platelet inhibition with aspirin therapy. rs12566888, which is in linkage disequilibrium with rs12041331, was associated with platelet aggregation phenotypes but to a lesser extent. In the EA families, the burden of PEAR1 missense variants was associated with platelet aggregation after aspirin therapy when the platelets were stimulated with epinephrine (p = 0.0009) and collagen (p = 0.03). In AAs, the burden of PEAR1 missense variants was associated, to a lesser degree, with platelet aggregation in response to epinephrine (p = 0.02) and ADP (p = 0.04).

Our study confirmed that the GWAS-identified variant, rs12041331, is the strongest variant associated with platelet aggregation both at baseline and after aspirin therapy in our GeneSTAR families in both races. We identified additional association of rare missense variants in PEAR1 with platelet aggregation following aspirin therapy. However, we observed a racial difference in the contribution of these rare variants to the platelet aggregation, most likely due to higher residual missing heritability of platelet aggregation after accounting for rs12041331 in the EAs compared to AAs.     

Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID

Viruses can create complex genetic populations within a host, and deep sequencing technologies allow extensive sampling of these populations. Limitations of these technologies, however, potentially bias this sampling, particularly when a PCR step precedes the sequencing protocol. Typically, an unknown number of templates are used in initiating the PCR amplification, and this can lead to unrecognized sequence resampling creating apparent homogeneity; also, PCR-mediated recombination can disrupt linkage, and differential amplification can skew allele frequency. Finally, misincorporation of nucleotides during PCR and errors during the sequencing protocol can inflate diversity. We have solved these problems by including a random sequence tag in the initial primer such that each template receives a unique Primer ID. After sequencing, repeated identification of a Primer ID reveals sequence resampling. These resampled sequences are then used to create an accurate consensus sequence for each template, correcting for recombination, allelic skewing, and misincorporation/sequencing errors. The resulting population of consensus sequences directly represents the initial sampled templates. We applied this approach to the HIV-1 protease (pro) gene to view the distribution of sequence variation of a complex viral population within a host. We identified major and minor polymorphisms at coding and noncoding positions. In addition, we observed dynamic genetic changes within the population during intermittent drug exposure, including the emergence of multiple resistant alleles. These results provide an unprecedented view of a complex viral population in the absence of PCR resampling.     

Spanish Guidelines for the use of targeted deep sequencing in myelodysplastic syndromes and chronic myelomonocytic leukaemia

The landscape of medical sequencing has rapidly changed with the evolution of next generation sequencing (NGS). These technologies have contributed to the molecular characterization of the myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML), through the identification of recurrent gene mutations, which are present in >80% of patients. These mutations contribute to a better classification and risk stratification of the patients. Currently, clinical laboratories include NGS genomic analyses in their routine clinical practice, in an effort to personalize the diagnosis, prognosis and treatment of MDS and CMML. NGS technologies have reduced the cost of large-scale sequencing, but there are additional challenges involving the clinical validation of these technologies, as continuous advances are constantly being made. In this context, it is of major importance to standardize the generation, analysis, clinical interpretation and reporting of NGS data. To that end, the Spanish MDS Group (GESMD) has expanded the present set of guidelines, aiming to establish common quality standards for the adequate implementation of NGS and clinical interpretation of the results, hoping that this effort will ultimately contribute to the benefit of patients with myeloid malignancies.