High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

Variations in 16S rRNA-based microbiome profiling between pyrosequencing runs and between pyrosequencing facilities

Pyrosequencing of 16S rRNA gene amplicons on the 454 FLX Titanium platform has been widely used to analyze microbiomes in various environments. However, different results may stem from variations among sequencing runs or among sequencing facilities. This study aimed to evaluate these variations between different pyrosequencing runs by sequencing 16S rRNA gene amplicon libraries generated from three sets of rumen samples twice each on the 454 FLX Titanium system at two independent sequencing facilities. Similar relative abundances were found for predominant taxa represented by large numbers of sequence reads but not for minor taxa represented by small numbers of sequence reads. The two sequencing facilities revealed different bacterial profiles with respect to both predominant taxa and minor taxa, including the most predominant genus Prevotella, the family Lachnospiraceae, and the phylum Proteobacteria. Differences in primers used to generate amplicon libraries may be a major source of variations in microbiome profiling. Because different primers and regions of 16S rRNA genes are often used by different researchers, significant variations likely exist among studies. Quantitative interpretation for relative abundance of taxa, especially minor taxa, from prevalence of sequence reads and comparisons of results from different studies should be done with caution.     

Parallel tagged sequencing on the 454 platform

Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplification or gel purification steps. The method relies on attaching sample-specific barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-five 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.     

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method

Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (相似文献   

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.     

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA

Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.     

High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.     

The Neandertal genome and ancient DNA authenticity

Recent advances in high‐thoughput DNA sequencing have made genome‐scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large‐scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot‐strap’ approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired.     

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics

BACKGROUND: Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. METHODOLOGY/PRINCIPALS FINDINGS: To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. CONCLUSIONS: We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5' and 3' ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

The antibody mining toolbox

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

High-throughput microsatellite marker development in two sparid species and verification of their transferability in the family Sparidae

Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.     

Linear amplification for deep sequencing

Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.     

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.     

Analysis of the Cystic Fibrosis Lung Microbiota via Serial Illumina Sequencing of Bacterial 16S rRNA Hypervariable Regions

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.     

Testing the limits of 454 pyrotag sequencing: reproducibility, quantitative assessment and comparison to T-RFLP fingerprinting of aquifer microbes

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.