Comparison of High-Throughput Sequencing for Phage Display Peptide Screening on Two Commercially Available Platforms

International Journal of Peptide Research and Therapeutics - Phage display is a widely used technique to screen peptide sequences for interaction with target biomolecules, such as proteins and...     

Comparison of Sample Preparation Methods Used for the Next-Generation Sequencing of Mycobacterium tuberculosis

The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.     

Comparison of Normalization Methods for Construction of Large,Multiplex Amplicon Pools for Next-Generation Sequencing

Constructing mixtures of tagged or bar-coded DNAs for sequencing is an important requirement for the efficient use of next-generation sequencers in applications where limited sequence data are required per sample. There are many applications in which next-generation sequencing can be used effectively to sequence large mixed samples; an example is the characterization of microbial communities where ≤1,000 sequences per samples are adequate to address research questions. Thus, it is possible to examine hundreds to thousands of samples per run on massively parallel next-generation sequencers. However, the cost savings for efficient utilization of sequence capacity is realized only if the production and management costs associated with construction of multiplex pools are also scalable. One critical step in multiplex pool construction is the normalization process, whereby equimolar amounts of each amplicon are mixed. Here we compare three approaches (spectroscopy, size-restricted spectroscopy, and quantitative binding) for normalization of large, multiplex amplicon pools for performance and efficiency. We found that the quantitative binding approach was superior and represents an efficient scalable process for construction of very large, multiplex pools with hundreds and perhaps thousands of individual amplicons included. We demonstrate the increased sequence diversity identified with higher throughput. Massively parallel sequencing can dramatically accelerate microbial ecology studies by allowing appropriate replication of sequence acquisition to account for temporal and spatial variations. Further, population studies to examine genetic variation, which require even lower levels of sequencing, should be possible where thousands of individual bar-coded amplicons are examined in parallel.Emergent technologies that generate DNA sequence data are designed primarily to perform resequencing projects at reasonable cost. The result is a substantial decrease in per base costs from traditional methods. However, these next-generation platforms do not readily accommodate projects that require obtaining moderate amounts of sequence from large numbers of samples. These platforms also have per run costs that are significant and generally preclude large numbers of single-sample, nonmultiplexed runs. One example of research that is not readily supported is rRNA-directed metagenomics study of some human clinical samples or environmental rRNA analysis of samples from communities with low community diversity that require only thousands of sequences. Thus, strategies to utilize next-generation DNA sequencers efficiently for applications that require lower throughput are critical to capitalize on the efficiency and cost benefits of next-generation sequencing platforms.Directed metagenomics based on amplification of rRNA genes is an important tool to characterize microbial communities in various environmental and clinical settings. In diverse environmental samples, large numbers of sequences are required to fully characterize the microbial communities (15). However, a lower number of sequences is generally adequate to answer specific research questions. In addition, the levels of diversity in human clinical samples are usually lower than what is observed in environmental samples (for example, see reference 7).The Roche 454 genome sequencer system FLX pyrosequencer (which we will refer to as 454 FLX hereafter) is the most useful platform for rRNA-directed metagenomics because it currently provides the longest read lengths of any next-generation sequencing platform (1, 14). Computational analysis has shown that the 250-nucleotide read length (available from the 454 FLX-LR chemistry) is adequate for identification of bacteria if the amplified region is properly positioned within variable regions of the small-subunit rRNA (SSU-rRNA) gene (9, 10).In this study, we used the 454 FLX-LR genome sequencing platform and chemistry, which provides >400,000 sequences of ∼250 bp per run. After we conducted this study, a new reagent set (454 FLX-XLR titanium chemistry) was released, which further increases reads to >1,000,000 and read lengths to >400 bp (Roche). The 454 FLX platform dramatically reduces per base costs of obtaining sequence, and physical separation into between 2 and 16 lanes is available; this physical separation on the plate reduces sequencing output overall, up to 40% comparing 2 lanes versus 16 lanes. For applications where modest sequencing depth (∼1,000 sequences per sample) is adequate to address research questions, physical separation does not allow adequate sample multiplexing because even a 1/16 454 FLX-LR plate run is expected to produce ∼15,000 reads. Further, the utility of the platform as a screening tool at 16-plex is limited by cost per run.A solution to make next-generation sequencing economical for projects such as rRNA-directed metagenomics is to use bar-coded primers to multiplex amplicon pools so they can be sequenced together and computationally separated afterward (6). To successfully accomplish this strategy, precise normalization of the DNA concentrations of the individual amplicons in the multiplex pools is essential for effective multiplex sequencing when large numbers of pooled samples are sequenced in parallel. There are several potential methods available for normalizing concentrations of amplicons included in multiplex pools, but the relative and absolute performance of each approach has not been compared.In this study, we present a direct quantitative comparison of three available methods for amplicon pool normalization for downstream next-generation sequencing. The central goal of the study was to identify the most effective method for normalizing multiplex pools containing >100 individual amplicons. We evaluated each pooling approach by 454 sequencing and compared the observed frequencies of sequences from different pooled bar-coded amplicons. From these data, we determined the efficacy of each method based on the following factors: (i) how well normalized the sequences within the pool were, (ii) the proportion of samples failing to meet a minimum threshold of sequences per sample, and (iii) the overall efficiency (speed and labor required) of the process to multiplex samples.     

Comparison of Two Commercially Available Media for Detection of Bacteremia

An analysis of 3,795 positive blood cultures obtained from 1,718 patients in a 2.5-year evaluation of Tryptic Soy Broth (TSB) and Thiol Broth is reported. Isolation rates of Actinobacillus and Pseudomonas were significantly greater in TSB, whereas isolation rates of Streptococcus and Corynebacterium (aerobic and anaerobic) were significantly greater in Thiol. Otherwise, the two media were similar. Disregarding contaminants, anaerobic bacteria represented 11% of positive cultures and 20% of patients with bacteremia. Eleven per cent of the patients had polymicrobial bacteremia.     

基于高通量测序技术的微生物检测数据分析方法

高通量测序技术的发展正在逐渐改变诸多生物学领域的研究方法.为应对突发疫情以及新发未知微生物威胁的需求,微生物鉴定技术逐渐从传统的物理化学方法及核酸杂交等分子水平方法进一步走向利用无需培养的测序数据进行快速分析检测.随之而来的是对高通量数据分析在精度及速度的要求.基于高通量测序数据的微生物检测数据分析方法在近些年得到了快速的发展.本文分析了目前基于高通量测序数据的微生物检测数据分析方法,对其数据分析的处理流程和计算方法进行了研究,比较了各个微生物检测数据分析方法的特点及适用场景.最后结合本实验室工作总结微生物检测数据分析方法在实际应用中可能遇到的问题,希望对该应用领域的研究有一定的参考意义.     

Comparative Studies of Copy Number Variation Detection Methods for Next-Generation Sequencing Technologies

Copy number variation (CNV) has played an important role in studies of susceptibility or resistance to complex diseases. Traditional methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution of genomic regions. Following the emergence of next generation sequencing (NGS) technologies, CNV detection methods based on the short read data have recently been developed. However, due to the relatively young age of the procedures, their performance is not fully understood. To help investigators choose suitable methods to detect CNVs, comparative studies are needed. We compared six publicly available CNV detection methods: CNV-seq, FREEC, readDepth, CNVnator, SegSeq and event-wise testing (EWT). They are evaluated both on simulated and real data with different experiment settings. The receiver operating characteristic (ROC) curve is employed to demonstrate the detection performance in terms of sensitivity and specificity, box plot is employed to compare their performances in terms of breakpoint and copy number estimation, Venn diagram is employed to show the consistency among these methods, and F-score is employed to show the overlapping quality of detected CNVs. The computational demands are also studied. The results of our work provide a comprehensive evaluation on the performances of the selected CNV detection methods, which will help biological investigators choose the best possible method.     

Multiplex Target Enrichment Using DNA Indexing for Ultra-High Throughput SNP Detection

Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.     

Next-Generation Sequencing of Aquatic Oligochaetes: Comparison of Experimental Communities

Aquatic oligochaetes are a common group of freshwater benthic invertebrates known to be very sensitive to environmental changes and currently used as bioindicators in some countries. However, more extensive application of oligochaetes for assessing the ecological quality of sediments in watercourses and lakes would require overcoming the difficulties related to morphology-based identification of oligochaetes species. This study tested the Next-Generation Sequencing (NGS) of a standard cytochrome c oxydase I (COI) barcode as a tool for the rapid assessment of oligochaete diversity in environmental samples, based on mixed specimen samples. To know the composition of each sample we Sanger sequenced every specimen present in these samples. Our study showed that a large majority of OTUs (Operational Taxonomic Unit) could be detected by NGS analyses. We also observed congruence between the NGS and specimen abundance data for several but not all OTUs. Because the differences in sequence abundance data were consistent across samples, we exploited these variations to empirically design correction factors. We showed that such factors increased the congruence between the values of oligochaetes-based indices inferred from the NGS and the Sanger-sequenced specimen data. The validation of these correction factors by further experimental studies will be needed for the adaptation and use of NGS technology in biomonitoring studies based on oligochaete communities.     

Comprehensive Mutation Analysis for Congenital Muscular Dystrophy: A Clinical PCR-Based Enrichment and Next-Generation Sequencing Panel

The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test''s appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel''s limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown.     

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.     

Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.     

Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing

The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies.     

Evaluation of Two Commercially Available Media for Detection of Bacteremia

Analysis of the results of 13,162 blood cultures during a 9-month interval has shown that Pseudomonas aeruginosa statistically was recovered more frequently from Trypticase soy broth (TSB) than from Thioglycollate-135C and that contaminants, including Staphylococcus epidermidis and aerobic and anaerobic Corynebacterium species, were isolated with statistically greater frequency from Thioglycollate-135C than from TSB. No other statistically significant differences were found.     

Development of Genetic Markers in Eucalyptus Species by Target Enrichment and Exome Sequencing

The advent of next-generation sequencing has facilitated large-scale discovery, validation and assessment of genetic markers for high density genotyping. The present study was undertaken to identify markers in genes supposedly related to wood property traits in three Eucalyptus species. Ninety four genes involved in xylogenesis were selected for hybridization probe based nuclear genomic DNA target enrichment and exome sequencing. Genomic DNA was isolated from the leaf tissues and used for on-array probe hybridization followed by Illumina sequencing. The raw sequence reads were trimmed and high-quality reads were mapped to the E. grandis reference sequence and the presence of single nucleotide variants (SNVs) and insertions/ deletions (InDels) were identified across the three species. The average read coverage was 216X and a total of 2294 SNVs and 479 InDels were discovered in E. camaldulensis, 2383 SNVs and 518 InDels in E. tereticornis, and 1228 SNVs and 409 InDels in E. grandis. Additionally, SNV calling and InDel detection were conducted in pair-wise comparisons of E. tereticornis vs. E. grandis, E. camaldulensis vs. E. tereticornis and E. camaldulensis vs. E. grandis. This study presents an efficient and high throughput method on development of genetic markers for family– based QTL and association analysis in Eucalyptus.     

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis(PGD) of patient’s embryos by next-generation sequencing(NGS) is dependent on efficient whole genome amplification(WGA) of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA(15 pg) and single cells, we showed that the two PCR-based WGA systems Sure Plex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification(MDA) system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing(CNV-Seq) was applied to single cell WGA products derived by either Sure Plex or MALBAC amplification, we showed that known disease CNVs in the range of 3e15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either Sure Plex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient’s cohort for uterine transplantation.