A Protein Deep Sequencing Evaluation of Metastatic Melanoma Tissues

Malignant melanoma has the highest increase of incidence of malignancies in the western world. In early stages, front line therapy is surgical excision of the primary tumor. Metastatic disease has very limited possibilities for cure. Recently, several protein kinase inhibitors and immune modifiers have shown promising clinical results but drug resistance in metastasized melanoma remains a major problem. The need for routine clinical biomarkers to follow disease progression and treatment efficacy is high. The aim of the present study was to build a protein sequence database in metastatic melanoma, searching for novel, relevant biomarkers. Ten lymph node metastases (South-Swedish Malignant Melanoma Biobank) were subjected to global protein expression analysis using two proteomics approaches (with/without orthogonal fractionation). Fractionation produced higher numbers of protein identifications (4284). Combining both methods, 5326 unique proteins were identified (2641 proteins overlapping). Deep mining proteomics may contribute to the discovery of novel biomarkers for metastatic melanoma, for example dividing the samples into two metastatic melanoma “genomic subtypes”, (“pigmentation” and “high immune”) revealed several proteins showing differential levels of expression. In conclusion, the present study provides an initial version of a metastatic melanoma protein sequence database producing a total of more than 5000 unique protein identifications. The raw data have been deposited to the ProteomeXchange with identifiers PXD001724 and PXD001725.     

Identification and Characterization of MicroRNAs in Pearl Oyster Pinctada martensii by Solexa Deep Sequencing

MicroRNAs (miRNAs) are short-nucleotide RNA molecules that function as negative regulators of gene expression in various organisms. However, miRNAs of Pinctada martensii have not been reported yet. P. martensii is one of the main species cultured for marine pearl production in China and Japan. In order to obtain the repertoire of miRNAs in P. martensii, we constructed and sequenced small RNA libraries prepared from P. martensii by Solexa deep sequencing technology and got a total of 27,479,838 reads representing 3,176,630 distinct sequences. After removing tRNAs, rRNAs, snRNAs, and snoRNAs, 10,596,306 miRNA reads representing 18,050 distinct miRNA reads were obtained. Based on sequence similarity and hairpin structure prediction, 258 P. martensii miRNAs (pm-miRNA) were identified. Among these pm-miRNAs, 205 were conserved across the species, whereas 53 were specific for P. martensii. The 3′ end sequence of U6 snRNA was obtained from P. martensii by 3′ rapid amplification of cDNA end PCR reaction and sequence-directed cloning. Eight conserved pm-miRNAs and two novel pm-miRNAs were validated by stem-loop quantitative real-time PCR with U6 snRNA as an internal reference gene. pm-miRNAs and the reported biomineralization-related genes were subjected to target analysis by using target prediction tools. Some of the pm-miRNAs, such as miR-2305 and miR-0046, were predicted to participate in biomineralization by regulating the biomineralization-related genes. Thus, this study demonstrated a large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation.     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

Deep Characterization of the Human Antibody Response to Natural Infection Using Longitudinal Immune Repertoire Sequencing

1. Download : Download high-res image (141KB)
2. Download : Download full-size image

Highlights

• •Longitudinal monitoring of B cell subsets shows baseline antibody gene expression.
• •A single, given CDR3 sequence can arise from more than one VJ gene combination.
• •A healthy individual's V gene usage is stable irrespective of infection and subset.
• •Surprisingly, class-switched antibodies can occur early in human B cell development.

     

Deep Sequencing and Ecological Characterization of Gut Microbial Communities of Diverse Bumble Bee Species

Gut bacterial communities of bumble bees are correlated with defense against pathogens. Further understanding this host-microbe association is vitally important as bumble bees are currently experiencing global population declines, potentially due in part to emergent diseases. In this study, we used pyrosequencing and community fingerprinting (ARISA) to characterize the gut microbial communities of nine bumble species from across the Bombus phylogeny. Overall, we delimited 74 bacterial taxa (operational taxonomic units or OTUs) belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, Actinobacteria, Flavobacteria and Alphaproteobacteria. Each bacterial community was taxonomically simple, containing an average of 1.9 common (relative abundance per sample > 5%) bacterial OTUs. The most abundant and prevalent (occurring in 92% of the samples) bacterial OTU, based on 16S rRNA sequences, closely matched that of the previously described Betaproteobacteria species Snodgrassella alvi. Bacteria that were first described in bee-related external environments dominated a number of gut bacterial communities, suggesting that they are not strictly dependent on the internal gut environment. The ARISA data showed a correlation between bacterial community structures and the geographic locations where the bees were sampled, suggesting that at least a subset of the bacterial species may be transmitted environmentally. Using light and fluorescent microscopy, we demonstrated that the gut bacteria form a biofilm on the internal epithelial surface of the ileum, corroborating results obtained from Apis mellifera.     

HIV-1 Quasispecies Delineation by Tag Linkage Deep Sequencing

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼0.005% to ∼0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.     

Hepatitis C Virus Transmission Bottlenecks Analyzed by Deep Sequencing

Hepatitis C virus (HCV) replication in infected patients produces large and diverse viral populations, which give rise to drug-resistant and immune escape variants. Here, we analyzed HCV populations during transmission and diversification in longitudinal and cross-sectional samples using 454/Roche pyrosequencing, in total analyzing 174,185 sequence reads. To sample diversity, four locations in the HCV genome were analyzed, ranging from high diversity (the envelope hypervariable region 1 [HVR1]) to almost no diversity (the 5′ untranslated region [UTR]). For three longitudinal samples for which early time points were available, we found that only 1 to 4 viral variants were present, suggesting that productive infection was initiated by a very small number of HCV particles. Sequence diversity accumulated subsequently, with the 5′ UTR showing almost no diversification while the envelope HVR1 showed >100 variants in some subjects. Calculation of the transmission probability for only a single variant, taking into account the measured population structure within patients, confirmed initial infection by one or a few viral particles. These findings provide the most detailed sequence-based analysis of HCV transmission bottlenecks to date. The analytical methods described here are broadly applicable to studies of viral diversity using deep sequencing.Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus of the flavivirus family. HCV infects ∼170 million people worldwide with a high rate of persistence (1, 2) and is a major etiological agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The current standard of therapy is the combined use of pegylated alpha interferon (IFN-α) and ribavirin (9), although there are substantial limitations due to toxicity and resistance profiles (47). Recent development of various small-molecule inhibitors that specifically target HCV offer some promise (13), but challenges still remain because the size and diversity of viral populations promote rapid development of drug resistance (28, 42). In an infected individual, serum HCV RNA levels can reach 10 to 100 million IU/ml (40). The viral RNA polymerase is estimated to make 1 error per 10,000 to 100,000 bp copied (22), but the viral genome is only 9,600 bases, resulting in diversification of the viral population, so that most viral genomes differ in sequence from the population consensus (16, 20, 21). Thus, when antiviral pressure is exerted on a viral population, sequence variants with reduced sensitivity may expand in the presence of the selective pressure (30, 41) and cause resistance (37). Consistent with this, differential sequence diversity in HCV populations has been linked to clinical outcome (7, 8).The size and complexity of HCV populations has made their analysis challenging. However, new deep-sequencing and bioinformatics methods are well suited to analyzing this problem. Using the 454/Roche technology, it is possible to generate more than 10⁸ bases of DNA sequence in a single 1-day run, albeit in fragments 200 to 500 bases in length (24). In addition, many samples can be multiplexed in single experiments using DNA barcodes introduced in amplification primers to tag each sample (3, 12, 45, 46), allowing many viral sequences to be characterized in a single experiment.Here, we analyze HCV diversity by pyrosequencing a series of representative viral regions contained within PCR amplicons, and we use methods from the environmental microbiology field for data processing and analysis. In both virology and environmental microbiology, populations of interest commonly consist of many related but nonidentical sequences (e.g., viral lineages with related sequences or bacteria harboring related 16S rRNA gene sequences). Assembly of short pyrosequence reads into longer scaffolds is quite difficult in such a setting, because the related sequences present in the population can be assembled in many different ways. Complicated data-processing methods yield at best complex probabilistic models of variants likely to be present in the population (6). For this reason, in studies of bacterial 16S DNA from uncultured communities, many groups have used simplified analysis of single 16S amplicons that query short regions of the 16S rRNA gene (5, 11, 14, 23, 25, 38, 44). Extensive simulations and practical applications show that analysis of such “sequence tags” can disclose biologically meaningful clusters and gradients in collections of samples. Here, we apply a similar approach, using sequence tags for several regions of the HCV genome. This approach has the disadvantage of losing linkage information between amplicons, but it does allow the efficient analysis of large numbers of viral variants over many samples.A major challenge, however, is distinguishing variations authentically present in viral populations from artifactual mutations introduced as a result of the isolation procedure or sequencing error. Sequence recovery involves PCR steps that can result in base pair substitutions or artifactual chimera formation. The 454/Roche method, like any sequencing method, has a characteristic error rate and particularly elevated error rates at homopolymer runs (24). In this study, we took advantage of improved methods for error control using the PyroNoise program of Quince and colleagues, which was first used for analysis of 16S rRNA gene sequences (29). The PyroNoise program preclusters the raw light intensity data generated during pyrosequencing by the 454/Roche method, which removes most homopolymer errors. In reconstruction experiments, Quince and colleagues showed that 454/Roche sequence analysis of artificially constructed mock 16S rRNA gene communities yielded greatly inflated numbers of sequence types due to error, but preclustering using PyroNoise reduced the diversity to values much closer to the correct value. Here, we used a two-stage clustering method to remove noise. In the first stage, raw light intensity data were preclustered with PyroNoise (29); then, in the second stage, after interpretation of the sequence as base calls, sequences were clustered at 98.5% identity. The second step allowed us to take advantage of redundancy in the reads to improve sequence quality, though distinguishing genuine low-level variations in the viral populations from error is a challenge.We determined 174,185 high-quality HCV sequence reads to characterize (i) longitudinal variation in HCV populations following transmission, (ii) differences in HCV variation between HCV-monoinfected and HIV-HCV-coinfected subjects, and (iii) variation in a control HCV genome cloned in a bacterial plasmid to quantify variation arising during the isolation and analytical procedure. We developed amplicons to characterize four regions of the HCV genome (Fig. ​(Fig.1A)1A) and found that HCV sequence diversity ranged from almost nonexistent to extreme depending on the region of the viral genome studied. Using the deep-sequencing data, we estimate that only one or a few viral variants seeded initial infection, but after that, viral variants could expand to >100 in a single individual. Thus, these data specify the numbers of particles seeding productive infection and provide a general framework for the use of deep-sequencing data to characterize the structures of viral populations.Open in a separate windowFIG. 1.HCV genome and characteristics of three subjects studied longitudinally during acute HCV infection. (A) The HCV genome and the positions of amplicons studied. The amplicons are numbered 1 to 4 from left to right, and a letter is used to indicate the direction of sequence determination. For the E1E2 HVR1 (3) and E2 (4) amplicons, two slightly different primers were used in each direction in an effort to maximize the diversity of recovered sequence variants, and these are indicated by the two bars. (B) HCV load and ALT levels for patients 1 to 3 during acute HCV infection. The x axis shows the number of weeks after clinical presentation, which for these patients was close in time to initial infection. Further patient characteristics were as follows: patient 1, injecting drug user, anti-HCV negative on 18 June 2001, first ALT flare (ALT, 677) on 6 July 2001, anti-HCV positive on 11 October 2001; patient 2, possible medical exposure, anti-HCV negative on 16 May 2001, initial ALT flare (ALT, 467) on 9 January 2004, anti-HCV positive on 22 April 2004; patient 3, injecting drug user, anti-HCV negative on 31 January 2006 (slightly abnormal ALT, 73), initial ALT flare (ALT, 640) on 10 April 2006, anti-HCV positive on 11 April 2006.     

Shaping of Human Germline IgH Repertoires Revealed by Deep Sequencing

To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。