An improved chloroplast DNA extraction procedure for whole plastid genome sequencing

Background

Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/Principal Findings

We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.

Conclusion

Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

The complete chloroplast genome of tall fescue (Lolium arundinaceum; Poaceae) and comparison of whole plastomes from the family Poaceae

In this paper, we describe the complete chloroplast genome of Lolium arundinaceum. This sequence is the culmination of a long-term project completed by >400 undergraduates who took general genetics at Middle Tennessee State University from 2004-2007. It was undertaken in an attempt to introduce these students to an open-ended experiential/exploratory lesson to produce and analyze novel data. The data they produced should provide the necessary information for both phylogenetic comparisons and plastome engineering of tall fescue. The fescue plastome (GenBank FJ466687) is 136048 bp with a typical quadripartite structure and a gene order similar to other grasses; 56% of the plastome is coding region comprised of 75 protein-coding genes, 29 tRNAs, four rRNAs, and one hypothetical coding region (ycf). Comparisons of Poaceae plastomes reveal size differences between the PACC (subfamilies Panicoideae, Arundinoideae, Centothecoideae, and Chloridoideae) and BOP (subfamilies Bambusoideae, Oryzoideae, and Pooideae) clades. Alignment analysis suggests that several potentially conserved large deletions in previously identified intergenic length polymorphic regions are responsible for the majority of the size discrepancy. Phylogenetic analysis using whole plastome data suggests that fescue closely aligns with Lolium perenne. Some unique features as well as phylogenetic branch length calculations, however, suggest that a number of changes have occurred since these species diverged.     

Phylogenetic relationships in Chinese oaks (Fagaceae,Quercus): Evidence from plastid genome using low-coverage whole genome sequencing

China is a second center of oak diversity but with less intensively systematic studies. Here, with 49 species representing all four sections in China, we firstly gave insight into the comprehensive phylogenetic relationships of Chinese oaks based on 54 complete plastid genomes. Our results recovered a robust phylogenetic framework and provided strong support for most nodes. The phylogenetic tree supported Quercus section Ilex as not monophyletic, in which Quercus section Cyclobalanopsis and Quercus section Cerris were nested. Most likely, incomplete lineage sorting and/or introgression among ancestral lineages in these three sections resulted in this complex pattern. The current distribution, diversification and molecular differentiation of Q. sect. Ilex in China are likely consequences of local adaptation to the geographic and paleoclimatic changes, which were driven by the uplift of Tibetan Plateau, the Hengduan Mountains and the Himalayas.     

Development and testing of novel chloroplast microsatellite markers for Lolium perenne and other grasses (Poaceae) from de novo sequencing and in silico sequences

Twelve primers to amplify microsatellite markers from the chloroplast genome of Lolium perenne were designed and optimized using de novo sequencing and in silico sequences. With one exception, each locus was polymorphic with a range from two to nine alleles in L. perenne. The newly developed primer pairs cross‐amplified in different species of Lolium and in 50 other grass species representing nine grass subfamilies.     

Long insert whole genome sequencing for copy number variant and translocation detection

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.     

Phylogenetic relationships in Southern African chloridoid grasses (Poaceae) based on nuclear and chloroplast sequence data

Phylogenetic relationships in southern African members of chloridoid grasses were investigated using DNA sequences from the chloroplast trnL (UAA) 5’ exon‐ trnF (GAA) region and the nuclear ribosomal internal transcribed spacer regions. The two datasets were analysed separately before being combined into a matrix of 50 specimens, representing 38 species. The congruence between the individual data sets was assessed in a conditional combination approach and the congruent data sets were then combined into a single data set. In this analysis, the chloridoid grasses were monophyletic and two large groups, corresponding to the tribes Eragrostideae and Cynodonteae, were polyphyletic; Eragrostis, the largest genus in the subfamily, was polyphyletic. Otherwise, high support levels were found at species and generic level.     

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis

Purpose of work  

A simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required.     

Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA

Besides their use in mRNA expression profiling, oligonucleotide microarrays have also been applied to single-nucleotide polymorphism (SNP) and loss of heterozygosity (LOH) or allelic imbalance studies. In this report, we evaluate the reliability of using whole genome amplified DNA for analysis with an oligonucleotide microarray containing 11 560 SNPs to detect allelic imbalance and chromosomal copy number abnormalities. Whole genome SNP analyses were performed with DNA extracted from osteosarcoma tissues and patient-matched blood. SNP calls were then generated by Affymetrix® GeneChip® DNA Analysis Software. In two osteosarcoma cases, using unamplified DNA, we identified 793 and 1070 SNP loci with allelic imbalance, respectively. In a parallel experiment with amplified DNA, 78% and 83% of these SNP loci with allelic imbalance was detected. The average false-positive rate is 13.8%. Furthermore, using the Affymetrix® GeneChip® Chromosome Copy Number Tool to analyze the SNP array data, we were able to detect identical chromosomal regions with gain or loss in both amplified and unamplified DNA at cytoband resolution.     

Identification of SNP markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae) using RAD sequencing

Phylogenetic relationships among temperate species of bamboo are difficult to resolve, owing to both the challenge of detecting sufficiently variable markers and their polyploid history. Here, we use restriction site–associated DNA sequencing to identify candidate loci with fixed allelic differences segregating between and within two temperate species of bamboos: Arundinaria faberi and Yushania brevipaniculata. Approximately 27 million paired‐end sequencing reads were generated across four samples. From pooled data, we assembled 67 685 and 70 668 de novo contigs from partial overlap among paired‐end reads, with an average length of 240 and 241 bp for the two species, respectively, which were used to investigate functional classification of RAD tags in a blastx search. Analysed separately by population, we recovered 29 443 putatively orthologous RAD tags shared across the four sampled populations, containing 28 023 sequence variants, of which c. 13 000 are segregating between species, and c. 3000 segregating between populations within each species. Analyses based on these RAD tags yielded robust phylogenetic inferences, even with data set constructed from surprisingly few loci. This study illustrates the potential for reduced‐representation genome data to resolve difficult phylogenetic relationships in temperate bamboos.     

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.     

An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of "natural" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.     

High-throughput sequencing of six bamboo chloroplast genomes: phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae)

Background

Bambusoideae is the only subfamily that contains woody members in the grass family, Poaceae. In phylogenetic analyses, Bambusoideae, Pooideae and Ehrhartoideae formed the BEP clade, yet the internal relationships of this clade are controversial. The distinctive life history (infrequent flowering and predominance of asexual reproduction) of woody bamboos makes them an interesting but taxonomically difficult group. Phylogenetic analyses based on large DNA fragments could only provide a moderate resolution of woody bamboo relationships, although a robust phylogenetic tree is needed to elucidate their evolutionary history. Phylogenomics is an alternative choice for resolving difficult phylogenies.

Methodology/Principal Findings

Here we present the complete nucleotide sequences of six woody bamboo chloroplast (cp) genomes using Illumina sequencing. These genomes are similar to those of other grasses and rather conservative in evolution. We constructed a phylogeny of Poaceae from 24 complete cp genomes including 21 grass species. Within the BEP clade, we found strong support for a sister relationship between Bambusoideae and Pooideae. In a substantial improvement over prior studies, all six nodes within Bambusoideae were supported with ≥0.95 posterior probability from Bayesian inference and 5/6 nodes resolved with 100% bootstrap support in maximum parsimony and maximum likelihood analyses. We found that repeats in the cp genome could provide phylogenetic information, while caution is needed when using indels in phylogenetic analyses based on few selected genes. We also identified relatively rapidly evolving cp genome regions that have the potential to be used for further phylogenetic study in Bambusoideae.

Conclusions/Significance

The cp genome of Bambusoideae evolved slowly, and phylogenomics based on whole cp genome could be used to resolve major relationships within the subfamily. The difficulty in resolving the diversification among three clades of temperate woody bamboos, even with complete cp genome sequences, suggests that these lineages may have diverged very rapidly.     

A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants

Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening projects.     

The chloroplast genome of Anomochloa marantoidea (Anomochlooideae; Poaceae) comprises a mixture of grass-like and unique features

Features in the complete plastome of Anomochloa marantoidea (Poaceae) were investigated. This species is one of four of Anomochlooideae, the crown node of which diverged before those of any other grass subfamily. The plastome was sequenced from overlapping amplicons using previously designed primers. The plastome of A. marantoidea is 138412 bp long with a typical gene content for Poaceae. Five regions were examined in detail because of prior surveys that identified structural alterations among graminoid Poales. Anomochloa marantoidea was found to have an intron in rpoC1, unlike other Poaceae. The insertion region of rpoC2 is unusually short in A. marantoidea compared with those of other grasses, but with atypically long subrepeats. Both ycf1 and ycf2 are nonfunctional as is typical in grasses, but A. marantoidea has a uniquely long ψycf1. Finally, the rbcL-psaI spacer in A. marantoidea is atypically short with no evidence of the ψrpl23 locus found in all other Poaceae. Some of these features are of noteworthy dissimilarity between A. marantoidea and those crown grasses for which entire plastomes have been sequenced. Complete plastome sequences of other Anomochlooideae and outgroups will further advance our understanding of the evolutionary events in the plastome that accompanied graminoid diversification.     

An efficient and tunable parameter to improve variant calling for whole genome and exome sequencing data

Next generation sequencing (NGS) has traditionally been performed in various fields including agricultural to clinical and there are so many sequencing platforms available in order to obtain accurate and consistent results. However, these platforms showed amplification bias when facilitating variant calls in personal genomes. Here, we sequenced whole genomes and whole exomes from ten Korean individuals using Illumina and Ion Proton, respectively to find the vulnerability and accuracy of NGS platform in the GC rich/poor area. Overall, a total of 1013 Gb reads from Illumina and ~39.1 Gb reads from Ion Proton were analyzed using BWA-GATK variant calling pipeline. Furthermore, conjunction with the VQSR tool and detailed filtering strategies, we achieved high-quality variants. Finally, each of the ten variants from Illumina only, Ion Proton only, and intersection was selected for Sanger validation. The validation results revealed that Illumina platform showed higher accuracy than Ion Proton. The described filtering methods are advantageous for large population-based whole genome studies designed to identify common and rare variations associated with complex diseases.     

Characterization of chloroplast microsatellite loci from whole chloroplast genome of Camellia taliensis and their utilization for evaluating genetic diversity of Camellia reticulata (Theaceae)

This study characterized chloroplast microsatellite markers for Camellia reticulata, a famous ornamental and edible economic tree species only distributed in Southwestern China. Thirty-two chloroplast microsatellite markers were originally annotated in the whole chloroplast genome of Camellia taliensis, ten polymorphic microsatellite markers were tested in 96 individuals from four natural populations of C. reticulata. Alleles numbered from two to four, and average value of Shannon's Information index, Nei's gene diversity, total genetic diversity, genetic diversity within populations, gene differentiation coefficient and gene flow index were 0.408, 0.225, 0.217, 0.102, 0.530 and 0.443, respectively. Our results showed high genetic differentiation and limited gene flow among the studied populations, which may be explained by recent fragmentation of the remnant populations due to human destruction and disturbance of natural habitats of the species. The identified cpSSR markers will be useful for the future studies on population genetics, conservation biology and phylogeography of C. reticulata.     

Tool evaluation for the detection of variably sized indels from next generation whole genome and targeted sequencing data

Insertions and deletions (indels) in human genomes are associated with a wide range of phenotypes, including various clinical disorders. High-throughput, next generation sequencing (NGS) technologies enable the detection of short genetic variants, such as single nucleotide variants (SNVs) and indels. However, the variant calling accuracy for indels remains considerably lower than for SNVs. Here we present a comparative study of the performance of variant calling tools for indel calling, evaluated with a wide repertoire of NGS datasets. While there is no single optimal tool to suit all circumstances, our results demonstrate that the choice of variant calling tool greatly impacts the precision and recall of indel calling. Furthermore, to reliably detect indels, it is essential to choose NGS technologies that offer a long read length and high coverage coupled with specific variant calling tools.