Functionally relevant microsatellites in sugarcane unigenes

<p>Abstract</p> <p>Background</p> <p>Unigene sequences constitute a rich source of functionally relevant microsatellites. The present study was undertaken to mine the microsatellites in the available unigene sequences of sugarcane for understanding their constitution in the expressed genic component of its complex polyploid/aneuploid genome, assessing their functional significance <it>in silico</it>, determining the extent of allelic diversity at the microsatellite loci and for evaluating their utility in large-scale genotyping applications in sugarcane.</p> <p>Results</p> <p>The average frequency of perfect microsatellite was 1/10.9 kb, while it was 1/44.3 kb for the long and hypervariable class I repeats. GC-rich trinucleotides coding for alanine and the GA-rich dinucleotides were the most abundant microsatellite classes. Out of 15,594 unigenes mined in the study, 767 contained microsatellite repeats and for 672 of these putative functions were determined <it>in silico</it>. The microsatellite repeats were found in the functional domains of proteins encoded by 364 unigenes. Its significance was assessed by establishing the structure-function relationship for the beta-amylase and protein kinase encoding unigenes having repeats in the catalytic domains. A total of 726 allelic variants (7.42 alleles per locus) with different repeat lengths were captured precisely for a set of 47 fluorescent dye labeled primers in 36 sugarcane genotypes and five cereal species using the automated fragment analysis system, which suggested the utility of designed primers for rapid, large-scale and high-throughput genotyping applications in sugarcane. Pair-wise similarity ranging from 0.33 to 0.84 with an average of 0.40 revealed a broad genetic base of the Indian varieties in respect of functionally relevant regions of the large and complex sugarcane genome.</p> <p>Conclusion</p> <p>Microsatellite repeats were present in 4.92% of sugarcane unigenes, for most (87.6%) of which functions were determined <it>in silico</it>. High level of allelic diversity in repeats including those present in the functional domains of proteins encoded by the unigenes demonstrated their use in assay of useful variation in the genic component of complex polyploid sugarcane genome.</p

The first draft of the pigeonpea genome sequence

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety ‘Asha’. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable ‘Arhar’ simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13562-011-0088-8) contains supplementary material, which is available to authorized users

High quality single amplicon sequencing method for illumina platforms using ‘N’ (0-10) spacer primer pool without PhiX spik-in

AbstractIllumina sequencing platform requires base diversity in initial 11 cycles for efficient cluster identification and color matrix estimation. This limitation yields low quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but overall reduces the number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms we developed high throughput single amplicon sequencing method by introducing ‘N’ spacers in target gene amplification primers that are pooled for simple handling. We evaluated the efficiency of ‘N’ spacer primers by targeting bacterial 16S V3-V4 region, demonstrating heterogonous base library construction. Addition of ‘N’ spacer causes sequencing frame shift at every base that leads to base diversity and produces heterogenous high quality reads within single amplicon library. We have written a python script “MetReTrim” to trim the heterogenous ‘N’ spacers from the pre-processed reads. This method terminates the need for PhiX spike-in and allows for multiplexing of multiple samples, greatly reducing the overall cost and yields improved sequence quality.</jats:p

A case of sinus venosus atrial septal defect misdiagnosed as primary pulmonary hypertension

AbstractWe present a case of sinus venosus atrial septal defect in a patient who was previously diagnosed as having primary pulmonary hypertension in a tertiary care center. Our findings are based on 2-dimensional trans-thoracic echocardiography, chest X–ray and surface electrocardiogram. A 26-year-old man, previously diagnosed as a case of primary pulmonary hypertension, presented to the emergency department (ED) with chest pain and breathlessness on exertion. Cardiac biomarkers were within their normal ranges. Surface electrocardiogram showed right atrial and ventricular overload with right axis deviation. Chest imaging noted enlarged central pulmonary vascularity with bilateral plethoric lung fields.Trans-thoracic echocardiography showed a dilated right atria and ventricle with severe tricuspid regurgitation and severe pulmonary artery hypertension with an intact atrial septum. Surprisingly, the transoesophageal echocardiogram revealed the presence of a sinus venous superior vena cava-type atrial septal defect with the right pulmonary vein draining into the right atria.In this full-text version, we present a more detailed discussion of sinus-venous atrial septal defect associated with partial anomalous pulmonary venous return that was wrongly diagnosed as a case of primary pulmonary hypertension in a tertiary care center

A case of sinus venosus atrial septal defect misdiagnosed as primary pulmonary hypertension

We present a case of sinus venosus atrial septal defect in a patient who was previously diagnosed as having primary pulmonary hypertension in a tertiary care center. Our findings are based on 2-dimensional trans-thoracic echocardiography, chest X–ray and surface electrocardiogram. A 26-year-old man, previously diagnosed as a case of primary pulmonary hypertension, presented to the emergency department (ED) with chest pain and breathlessness on exertion. Cardiac biomarkers were within their normal ranges. Surface electrocardiogram showed right atrial and ventricular overload with right axis deviation. Chest imaging noted enlarged central pulmonary vascularity with bilateral plethoric lung fields. Trans-thoracic echocardiography showed a dilated right atria and ventricle with severe tricuspid regurgitation and severe pulmonary artery hypertension with an intact atrial septum. Surprisingly, the transoesophageal echocardiogram revealed the presence of a sinus venous superior vena cava-type atrial septal defect with the right pulmonary vein draining into the right atria. In this full-text version, we present a more detailed discussion of sinus-venous atrial septal defect associated with partial anomalous pulmonary venous return that was wrongly diagnosed as a case of primary pulmonary hypertension in a tertiary care center

Optimized protocol for assay for transposase-accessible chromatin by sequencing (ATAC-seq) from Drosophila melanogaster brain tissue

Summary: Transposase-accessible chromatin by sequencing (ATAC-seq) has emerged as an advantageous technique to assess chromatin accessibility owing to the robustness of ''tagmentation'' process and a relatively faster library preparation. A comprehensive ATAC-seq protocol from Drosophila brain tissue is currently unavailable. Here, we have provided a detailed protocol of ATAC-seq assay from Drosophila brain tissue. Starting from dissection and transposition to amplification of libraries has been elaborated. Furthermore, a robust ATAC-seq analysis pipeline has been presented. The protocol can be easily adapted for other soft tissues. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes

miRNA-mediated silencing of R2R3 MYB transcription factor promotes the production of specific secondary metabolites in grapes.</jats:p

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

Abstract Background Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on illumina platforms, we developed a high throughput single amplicon sequencing method by introducing ‘N’ (0–10) spacers in target gene amplification primers that are pooled for simple handling. Result We evaluated the efficiency of ‘N’ (0–10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of ‘N’ (0–10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,“MetReTrim”, to trim the ‘N’ (0–10) spacers from the raw reads (https://github.com/Mohak91/MetReTrim). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We observed no difference between the communities represented by our method and standard illumina V3-V4 primer method. Conclusion This method eliminates the need for PhiX spike-in for single amplicon sequencing on illumina MiSeq platform. This allows for sequencing of more number of samples in a run and a reduction in the overall cost. Given that Illumina sequencing works on SBS chemistry irrespective of the platform (such as HiSeq, MiSeq, NextSeq, NovaSeq, etc.) we propose that this strategy of using ‘N’ (0–10) spacer-linked primer design can be adopted for generating high-quality single locus amplicon sequencing in a high throughput manner across the illumina platform subject to further validation. </jats:sec