Genomic resources of broomcorn millet: demonstration and application of a high-throughput BAC mapping pipeline

Background With high-efficient water-use and drought tolerance, broomcorn millet has emerged as a candidate for food security. To promote its research process for molecular breeding and functional research, a comprehensive genome resource is of great importance. Results Herein, we constructed a BAC library for broomcorn millet, generated BAC end sequences based on the clone-array pooled shotgun sequencing strategy and Illumina sequencing technology, and integrated BAC clones into genome by a novel pipeline for BAC end profiling. The BAC library consisted of 76,023 clones with an average insert length of 123.48 Kb, covering about 9.9-fold of the 850 Mb genome. Of 9216 clones tested using our pipeline, 8262 clones were mapped on the broomcorn millet cultivar longmi4 genome. These mapped clones covered 308 of the 829 gaps left by the genome. To our knowledge, this is the only BAC resource for broomcorn millet. Conclusions We constructed a high-quality BAC libraray for broomcorn millet and designed a novel pipeline for BAC end profiling. BAC clones can be browsed and obtained from our website (http://eightstarsbio.com/gresource/JBrowse-1.16.5/index.html). The high-quality BAC clones mapped on genome in this study will provide a powerful genomic resource for genome gap filling, complex segment sequencing, FISH, functional research and genetic engineering of broomcorn millet.

Genome-wide cell-free DNA termini in patients with cancer.

The structure, fragmentation pattern, length and terminal sequence of cell-free DNA (cfDNA) is under the influence of nucleases present in the blood. We hypothesized that differences in the diversity of bases at the end of cfDNA fragments can be leveraged on a genome-wide scale to enhance the sensitivity for detecting the presence of tumor signals in plasma. We surveyed the cfDNA termini in 572 plasma samples from 319 patients with 18 different cancer types using low-coverage whole genome sequencing. The fragment-end sequence and diversity were altered in all cancer types in comparison to 76 healthy controls. We converted the fragment end sequences into a quantitative metric and observed that this correlates with circulating tumor DNA tumor fraction (R = 0.58, p < 0.001, Spearman). Using these metrics, we were able to classify cancer samples from control at a low tumor content (AUROC of 91% at 1% tumor fraction) and shallow sequencing coverage (mean AUROC = 0.99 at >1M fragments). Combining fragment-end sequences and diversity using machine learning, we classified cancer from healthy controls (mean AUROC = 0.99, SD = 0.01). Using unsupervised clustering we showed that early-stage lung cancer (n = 13) can be classified from control or later stages based on fragment-end sequences. We observed that fragment-end sequences can be used for prognostication (hazard ratio: 0.49) and residual disease detection in resectable esophageal adenocarcinoma patients, moving fragmentomics toward a greater clinical implementation.

Gene identification and genome annotation in Caenorhabditis briggsae by high throughput 5' RNA end determination

The nematode Caenorhabditis briggsae is routinely used in comparative and evolutionary studies involving its well-known cousin C. elegans. The C. briggsae genome sequence has accelerated research by facilitating the generation of new resources, tools, and functional studies of genes. While substantial progress has been made in predicting genes and start sites, experimental evidence is still lacking in many cases. Here, we report an improved annotation of the C. briggsae genome using the Trans-spliced Exon Coupled RNA End Determination (TEC-RED) technique. In addition to identifying 5' ends of expressed genes, the technique has enabled the discovery of operons and paralogs. Application of TEC-RED yielded 10,243 unique 5' end sequences with matches in the C. briggsae genome. Of these, 6,395 were found to represent 4,252 unique genes along with 362 paralogs and 52 previously unknown exons. The method also identified 493 operons, including 334 that are fully supported by tags. Additionally, two SL1-type operons were discovered. Comparisons with C. elegans revealed that 40% of operons are conserved. Further, we identified 73 novel operons, including 12 that entirely lack orthologs in C. elegans. Among other results, we found that 14 genes are trans-spliced exclusively in C. briggsae compared with C. elegans. Altogether, the data presented here serves as a rich resource to aid biological studies involving C. briggsae. Additionally, this work demonstrates the use of TEC-RED for the first time in a non-elegans nematode and suggests that it could apply to other organisms with a trans-splicing reaction from spliced leader RNA.

Devising an efficient approach to determine the optimal sequence of from-to matrix

Sequencing is the most impact factor in many production areas, such as assembly lines, batch production, Travelling Salesman Problem (TSP), product sequences, process sequences, etc. The flow and analysis from one item to another can be presented by the square matrix in which the number of rows or columns is equal to the number of manipulated items, this special matrix form is called “From-To matrix”. The matrix suffers from many drawbacks when it is applied to determine the optimal sequences, such as the number of variables must be as small as possible, there is no flexibility to determine the start or the end sequence to find the best sequencing with some conditions. Also, there is no possibility to add relations to point a variable as wanted or prevented from the sequence. In this paper, we solve the From-To matrix by binary linear programming (BLP). The proposed BLP approach has been applied in Ur company to solve the From-To matrix. This company has a production line that can manufacture four products: A, B, C, and D, the setup time matrix is considered as From-To matrix and the goal of this company is to get an optimum sequence of products with minimum time. The solution of state transition of the From-To matrix using BLP can be formulated in the following five model cases according to transition requirement condition and desired: the first case gives all possible sequence items, the second case lists the sequence items when the first sequence is known, the third case lists the sequence items when the last sequence is known, the fourth case gives all possible sequence items with a condition that prevents occurring of an undesired sequence, and the fifth case gives all possible sequence items with the condition of a wanted occurring of the desired sequence. Furthermore, we found the optimum sequences for states by determining the start or end sequences, and also add the wanted relations or prevented. The mathematical formulas for the number of all sequences under some conditions are derived and proved

Genomic Resources of Broomcorn Millet: Demonstration and Application of a High-throughput BAC Mapping Pipeline

Background: With high-efficient water-use and drought tolerance, broomcorn millet has emerged as a candidate for food security. To promote its research process for molecular breeding and functional research, a comprehensive genome resource is of great importance. Results: Herein, we constructed a BAC library for broomcorn millet, generated BAC end sequences based on the clone-array pooled shotgun sequencing strategy and Illumina sequencing technology, and integrated BAC clones into genome by a novel pipeline for BAC end profiling. The BAC library is consisted of 76,023 clones with an average insert length of 123.48 Kb, covering about 9.9-fold of the 850 Mb genome. Of 9,216 clones tested using our pipeline, 8,262 clones were mapped on the broomcorn millet cultivar longmi4 genome. These mapped clones covered 308 of the 829 gaps left by the genome. To our knowledge, this is the only BAC resource for broomcorn millet.Conclusions: We constructed a high-quality BAC libraray for broomcorn millet and designed a novel pipeline for BAC end profiling. BAC clones can be browsed and obtained from our website (http://eightstarsbio.com/gresource/JBrowse-1.16.5/index.html). The high-quality BAC clones mapped on genome in this study will provide a powerful genomic resource for genome gap filling, complex segment sequencing, FISH, functional research, and genetic engineering of broomcorn millet.

The Integration Preference of Sleeping Beauty at Non-TA Site Is Related to the Transposon End Sequences

Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.

Genomic Resources of Broomcorn Millet: Demonstration and Application of a High-throughput BAC Mapping Pipeline

BackgroundWith high-efficient water-use and drought tolerance, broomcorn millet has emerged as candidate for food security. To promote its research process for molecular breeding and functional research, a comprehensive genome resources is of great important.ResultsHerein, we constructed the first BAC library for broomcorn millet, generated BAC end sequences and integrated BAC clones into genome by a novel pipeline for BAC end profiling depending on clone-array pooled shotgun sequencing strategy and Illumina sequencing technology. The BAC library is consisted of 76,023 clones with average insert length of 123.48 Kb, about 9.9x coverage of 850 Mb genome. Then, 8262 of 9216 clones were mapped on broomcorn millet cultivar longmi4 genome using our pipeline. Furthermore, we also extracted and assembled unmapped reads against longmi4 genome. A total of 135 deletion sequences, 64 specific sequences and some sample contamination sequences were identified.ConclusionsBAC clones in this library can be browsed and obtained from our website (http://flyinguineapig.com/gresource/JBrowse-1.16.5/index.html). This pipeline designed for BAC end profiling can greatly reduce the cost of acquiring BAC end sequences and shorten the period of the experiment compared with the Sanger sequencing method. These high-quality BAC clones related with genome in this study provide a useful and convinient genomic resource for genome gap filling, complex segment sequencing, FISH, functional research, and genetic engineering.

Cloning and preliminary verification of telomere-associated sequences in upland cotton

Telomeres are structures enriched in repetitive sequences at the end of chromosomes. In this study, using the telomere primer AA(CCCTAAA)3CCC for the single primer PCR, two DNA sequences were obtained from Gossypium hirsutum (Linnaeus, 1753) accession (acc.) TM-1. Sequence analysis showed that the two obtained sequences were all rich in A/T base, which was consistent with the characteristic of the telomere-associated sequence (TAS). They were designated as GhTAS1 and GhTAS2 respectively. GhTAS1 is 489 bp long, with 57.6% of A/T, and GhTAS2 is 539 bp long, with 63.9% of A/T. Fluorescence in situ hybridization results showed that both of the cloned TASs were located at the ends of the partial chromosomes of G. hirsutum, with the strong signals, which further confirmed that GhTAS1 and GhTAS2 were telomere-associated sequences including highly tandemly repetitive sequences. Results of blast against the assembled genome of G. hirsutum showed that GhTAS sequences may be missed on some assembled chromosomes. The results provide important evidence for the evaluation of the integrity of assembled chromosome end sequences, and will also contribute to the further perfection of the draft genomes of cotton.

The integration preference of Sleeping Beauty at non-TA site is related to the transposon end sequences

Background: Sleeping Beauty (SB) transposon had been thought to strictly integrate into TA dinucleotides. Recently, we found that SB also integrates into non-TA sites at a lower frequency. Here we performed further study on the non-TA integration of SB. Results: 1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines. 2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites. 3) The consensus sequence of the non-TA target sites only occur at the opposite side of the sequenced junction between transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations. 4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. When the transposon end sequence is mutated, the consensus sequences changed too. Conclusion: The interaction between the SB transposon end and genomic DNA may be involved in the target site selection of the SB integrations at non-TA sites.