Aquileo | Recent changes to wiki

Aquileo | Home modified by Jorge Duitama

Fri, 16 Aug 2024 15:16:10 -0000

--- v138
+++ v139
@@ -94,7 +94,7 @@
 4 [README](https://sourceforge.net/projects/ngsep/files/Library/README.txt/download) with a detailed description of all modules and parameters.
 5. See what's new in the [Change Log](https://sourceforge.net/projects/ngsep/files/Library/CHANGELOG/download).

-* [User manual for the new graphical interface (under construction)](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf)
+* [User manual for the new graphical interface (under construction)](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v5.0.0.pdf)

 * [User Manual of version 3](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download) for the legacy Eclipse plugin
 - - - - - -

Aquileo | Home modified by Jorge Duitama

Tue, 02 Jul 2024 21:31:05 -0000

--- v137
+++ v138
@@ -80,7 +80,7 @@

 #### Graphical interface : ####

-Since version 4 we built a new [graphical interface](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is under construction, but it can be accessed [here](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).
+Since version 4 we built a new [graphical interface](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface can be accessed [here](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v5.0.0.pdf).

 The Eclipse plugin will no longer be sustained. However, the plugin with the functionalities of NGSEP 3 is still [available](http://sourceforge.net/projects/ngsep/files/OnlyPlugin/). The package including Eclipse and an installer of bowtie2 is also [available](http://sourceforge.net/projects/ngsep/files/Eclipse%2BPlugin/). To update an existing installation, just download the plugin and follow the instructions in the [User Manual](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download).

Aquileo | Home modified by Jorge Duitama

Tue, 02 Jul 2024 21:26:31 -0000

--- v136
+++ v137
@@ -133,7 +133,7 @@
 NGSEP3: accurate variant calling across species and sequencing protocols.
 *Bioinformatics* 35(22): 4716–4723. http://doi.org/10.1093/bioinformatics/btz275

-***Transposable elements: ***
+***Transposable elements:***

 Our approach to map  known transposable elements to a genome assembly, based on minimizers can be found in Applications in Plant Sciences

@@ -155,7 +155,7 @@
 Structural variants in 3000 rice genomes.
 Genome Research 29: 870-880. http://doi.org/10.1101/gr.241240.118

-***TILLING: ***
+***TILLING:***

 Functionalities related to the TILLING experimental setup can be found in Frontiers in Genetics:

@@ -163,7 +163,7 @@
 Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.
 *Frontiers in Genetics*  12: 54. http://doi.org/10.3389/fgene.2021.624513

-***GBS pipelines: ***
+***GBS pipelines:***

 Further details on the pipeline built for reference-guided variants detection on Genotype-By-Sequencing (GBS) data can be found at BMC Genomics:

Aquileo | Home modified by Jorge Duitama

Fri, 26 Jan 2024 01:36:50 -0000

--- v135
+++ v136
@@ -94,7 +94,7 @@
 4 [README](https://sourceforge.net/projects/ngsep/files/Library/README.txt/download) with a detailed description of all modules and parameters.
 5. See what's new in the [Change Log](https://sourceforge.net/projects/ngsep/files/Library/CHANGELOG/download).

-* [User manual for the new graphical interface of version 4](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf)
+* [User manual for the new graphical interface (under construction)](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf)

 * [User Manual of version 3](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download) for the legacy Eclipse plugin
 - - - - - -

Aquileo | Home modified by Jorge Duitama

Fri, 26 Jan 2024 01:35:32 -0000

--- v134
+++ v135
@@ -21,9 +21,11 @@
 2.  Spectrum of k-mer abundances from fastq or fasta files 
 3. Raw reads error correction
 4. De-novo assembly of long reads
-5. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
-6. De-novo analysis of Genotype-by-sequencing (GBS) reads
-7. Alignment of raw reads to a reference genome
+5. **NEW** Sorting of genome assemblies by comparison to a close reference genome
+6. **NEW** Circularization of circular molecules in genome assemblies
+7. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
+8. De-novo analysis of Genotype-by-sequencing (GBS) reads
+9. Alignment of raw reads to a reference genome

 #### Variants discovery and genotyping ####

@@ -42,8 +44,8 @@
 2.  Filtering of transcriptome files in GFF3 format
 3.  Large scale alignment of two assembled and annotated genomes.
 4. Identification and clustering of orthologs and paralogs
-5. **NEW**  Library-guided annotation of transposable elements in a genome assembly.
-6. **NEW**  Masking of genome assemblies.
+5. Library-guided annotation of transposable elements in a genome assembly.
+6. Masking of genome assemblies.

 #### Variants (VCF) downstream analysis ####

@@ -78,7 +80,7 @@

 #### Graphical interface : ####

-A graphical interface for NGSEP 4 is  [available](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is under construction, but it can be accessed [here](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).
+Since version 4 we built a new [graphical interface](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is under construction, but it can be accessed [here](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).

 The Eclipse plugin will no longer be sustained. However, the plugin with the functionalities of NGSEP 3 is still [available](http://sourceforge.net/projects/ngsep/files/OnlyPlugin/). The package including Eclipse and an installer of bowtie2 is also [available](http://sourceforge.net/projects/ngsep/files/Eclipse%2BPlugin/). To update an existing installation, just download the plugin and follow the instructions in the [User Manual](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download).

@@ -131,6 +133,28 @@
 NGSEP3: accurate variant calling across species and sequencing protocols.
 *Bioinformatics* 35(22): 4716–4723. http://doi.org/10.1093/bioinformatics/btz275

+***Transposable elements: ***
+
+Our approach to map  known transposable elements to a genome assembly, based on minimizers can be found in Applications in Plant Sciences
+
+Gonzalez-García LN, Lozano-Arce D, Londoño JP, Guyot R and Duitama J. (2023).
+Efficient homology-based annotation of transposable elements using minimizers.
+*Applications in Plant Sciences* 11(4): e11520. http://doi.org/10.1002/aps3.11520
+
+***Structural variants detection:***
+
+For long reads, our approach based on the DBScan clustering algorithm can be found in GigaScience
+
+Gaitán N and Duitama J. (2024)
+A graph clustering algorithm for detection and genotyping of structural variants from long reads
+*GigaScience* 13: giad112. https://doi.org/10.1093/gigascience/giad112
+
+For short reads, since version 2.1.2, we implemented an algorithm to integrate paired-end and split-read analysis for detection of large indels. Benchmark experiments of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:
+
+Fuentes RR, Chebotarov D, Duitama J, Smith S, De la Hoz JF, Mohiyuddin M, et al. (2019).
+Structural variants in 3000 rice genomes.
+Genome Research 29: 870-880. http://doi.org/10.1101/gr.241240.118
+
 ***TILLING: ***

 Functionalities related to the TILLING experimental setup can be found in Frontiers in Genetics:
@@ -187,13 +211,6 @@
 Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection.
 *PLoS ONE* 10(4): e0124617. http://doi.org/10.1371/journal.pone.0124617

-***Structural variants detection:***
-
-Since version 2.1.2, we implemented a model to integrate paired-end and split-read analysis for detection of large indels. Benchmark experiments of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:
-
-Fuentes RR, Chebotarov D, Duitama J, Smith S, De la Hoz JF, Mohiyuddin M, et al. (2019).
-Structural variants in 3000 rice genomes.
-Genome Research 29: 870-880. http://doi.org/10.1101/gr.241240.118

 **NGSEP** is also supported by the following open source software packages:

Aquileo | Home modified by Jorge Duitama

Wed, 05 Apr 2023 19:02:23 -0000

--- v133
+++ v134
@@ -103,11 +103,17 @@
 - - - - - - -
 Citing
 ===
-The manuscript of NGSEP 4, focused on orthologs and genome alignment has been recently published at Molecular Ecology Resources:
-
-Tello D, Gonzalez-Garcia LN, Gomez J, et al. (2022).
+The manuscript of **NGSEP 4**, focused on orthologs and genome alignment is available at Molecular Ecology Resources:
+
+Tello D, Gonzalez-Garcia LN, Gomez J, et al. (2023).
 NGSEP 4: Efficient and accurate identification of orthogroups and whole-genome alignment.
-*Molecular Ecology Resources in press*. https://doi.org/10.1111/1755-0998.13737
+*Molecular Ecology Resources* 23(3): 712-724. https://doi.org/10.1111/1755-0998.13737
+
+The manuscript describing the new functionality of **NGSEP** for de-novo genome assembly of long reads is available at Life Science Alliance:
+
+Gonzalez-Garcia L, Guevara-Barrientos D, Lozano-Arce D et al. (2023).
+New algorithms for accurate and efficient de novo genome assembly from long DNA sequencing reads.
+*Life Science Alliance* 6(5): . http://doi.org/10.26508/lsa.202201719

 The manuscript with the description of the initial modules of **NGSEP** is available at Nucleic Acids research:

@@ -147,14 +153,6 @@
 Robust and efficient software for reference-free genomic diversity analysis of GBS data on diploid and polyploid species
 *Molecular Ecology Resources* 22(1): 439-454. http://doi.org/10.1101/2020.11.28.402131

-***Genome Assembly: ***
-
-A manuscript describing the algorithms for de-novo genome assembly of long reads is under review. In the mean time, the details of the algorithms are described in this preprint:
-
-Gonzalez-Garcia, L., Guevara-Barrientos D., Lozano-Arce D., *et al*.
-New algorithms for accurate and efficient de-novo genome assembly from long DNA sequencing reads. 
-*bioRxiv* 2022.08.30.505891. https://doi.org/10.1101/2022.08.30.505891 
-
 ***Molecular haplotyping:***

 Duitama J, McEwen GK, Huebsch T, Palczewski S, Schulz S, Verstrepen K, *et al*. (2011)
@@ -188,7 +186,6 @@
 Duitama J, Silva A, Sanabria Y, Cruz DF, Quintero C, Ballen C, et al. (2015)
 Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection.
 *PLoS ONE* 10(4): e0124617. http://doi.org/10.1371/journal.pone.0124617
-

 ***Structural variants detection:***

@@ -208,7 +205,103 @@
 - - - - - -
 Publications citing NGSEP
 ===
-Different modules of NGSEP have been used to perform bioinformatic analysis or as a baseline for development of novel tools in the following publications:
+Different modules of NGSEP have been used to perform bioinformatic analysis in more than 100 scientific publications:
+
+Amongi W, Nkalubo ST, Ochwo-Ssemakula M, et al. (2023).
+Genetic clustering, and diversity of African panel of released common bean genotypes and breeding lines.
+*Genetic Resources and Crop Evolution in press*. https://doi.org/10.1007/s10722-023-01559-y
+
+Vega M, Quintero-Correales C, Mastretta-Yanes A, et al. (2023).
+Multiple domestication events explain the origin of Gossypium hirsutum landraces in Mexico
+*Ecology and Evolution* 13(3): e9838
+
+Ethridge SR, Chandra S, Everman W, et al. (2023).
+Rapid evolution of competitive ability in giant foxtail (Setaria faberi) over 34 years.
+*Weed Science* 71(1): 59-68. http://doi.org/10.1017/wsc.2023.1
+
+De Souza Rodrigues Marinho J, Valdisser PAMR, Brondani C, et al. (2023).
+Molecular markers for assessing the inter- and intra-racial genetic diversity and structure of common bean.
+*Genetic Resources and Crop Evolution* 70: 263–279. https://doi.org/10.1007/s10722-022-01432-4
+
+Farek J, Hughes D, Salerno W, et al. (2023).
+xAtlas: scalable small variant calling across heterogeneous next-generation sequencing experiments.
+*GigaScience* 12: giac125. https://doi.org/10.1093/gigascience
+
+Kang H, An S-M, Park Y-J, et al. (2023).
+Population Genomics Study and Implications for the Conservation of Zabelia tyaihyonii Based on Genotyping-By-Sequencing.
+*Plants* 12(1):171. https://doi.org/10.3390/plants12010171 
+
+García Navarrete T, Arias C, Mukundi E, et al. (2022).
+Natural variation and improved genome annotation of the emerging biofuel crop field pennycress (Thlaspi arvense).
+*G3* 12(6): jkac084. https://doi.org/10.1093/g3journal/jkac084
+
+He T, Ye C, Zeng Q, et al. (2022).
+Genetic diversity and population structure of cultivated Dendrobium nobile Lindl. in southwest of China based on genotyping-by-sequencing.
+*Genetic Resources and Crop Evolution* 69: 2803–2818. https://doi.org/10.1007/s10722-022-01401-x
+
+Muli JK, Neondo JO, Kamau PK, et al. (2022).
+Genetic diversity and population structure of wild and cultivated Crotalaria species based on genotyping-by-sequencing.
+*PLoS ONE* 17(9): e0272955. https://doi.org/10.1371/journal.pone.0272955
+
+Lui C, Wang Y, Peng J, et al. (2022)
+High-quality genome assembly and pan-genome studies facilitate genetic discovery in mung bean and its improvement.
+*Plant Communications* 3 (6): 100352. https://doi.org/10.1016/j.xplc.2022.100352.
+
+Upadhyay P, Gupta M, Sra SK, et al. (2022).
+Genome wide association studies for acid phosphatase activity at varying phosphorous levels in Brassica juncea L.
+*Frontiers in Plant Sciences* 13:1056028. http://doi.org/10.3389/fpls.2022.1056028
+
+Barrera S, Teran JCBM, Lobaton JD, et al. (2022).
+Large genomic introgression blocks of Phaseolus parvifolius Freytag bean into the common bean enhance the crossability between tepary and common beans.
+*Plant direct* 6(12): e470. https://doi.org/10.1002/pld3.470
+
+Keller B, Ariza-Suarez D, Portilla-Benavides AE, et al. (2022).
+Improving Association Studies and Genomic Predictions for Climbing Beans With Data From Bush Bean Populations.
+*Frontiers in Plant Sciences* 13:830896. http://doi.org/10.3389/fpls.2022.830896
+
+Velez N, Vega-Vela N, Muñoz M, et al. (2022).
+Deciphering the Association among Pathogenicity, Production and Polymorphisms of Capsule/Melanin in Clinical Isolates of Cryptococcus neoformans var. grubii VNI.
+*Journal of Fungi* 8(3): 245. https://doi.org/10.3390/jof8030245
+
+Saballos A, Soler-Garzón A, Brooks M, et al. (2022).
+Multiple Genomic Regions Govern Tolerance to Sulfentrazone in Snap Bean (Phaseolus Vulgaris L.).
+*Frontiers in Agronomy* 4:869770. http://doi.org/10.3389/fagro.2022.869770
+
+Sadohara R, Izquierdo P, Couto Alves F. et al. (2022).
+The Phaseolus vulgaris L. Yellow Bean Collection: genetic diversity and characterization for cooking time.
+*Genetic Resources and Crop Evolution* 69: 1627–1648. https://doi.org/10.1007/s10722-021-01323-0
+
+Diaz S, Polania J, Ariza-Suarez D, et al. (2022).
+Genetic Correlation Between Fe and Zn Biofortification and Yield Components in a Common Bean (Phaseolus vulgaris L.).
+*Frontiers in Plant Science* 12:739033. http://doi.org/10.3389/fpls.2021.739033
+
+Souffriau B, Holt S, Hagman A, et al. (2022).
+Polygenic Analysis of Tolerance to Carbon Dioxide Inhibition of Isoamyl Acetate “Banana” Flavor Production in Yeast Reveals MDS3 as Major Causative Gene.
+*Applied and Environmental Microbiology* 88 (18): e0081422. http://doi.org/10.1128/aem.00814-22.
+
+Ariza-Suarez D, Keller B, Spescha A, et al. (2022).
+Genetic analysis of resistance to bean leaf crumple virus identifies a candidate LRR-RLK gene.
+*The plant journal* 114(1): 23-38. https://doi.org/10.1111/tpj.15810
+
+Stojiljkovic M, Claes A, Deparis Q, et al. (2022).
+Whole-Genome Transformation of Yeast Promotes Rare Host Mutations with a Single Causative SNP Enhancing Acetic Acid Tolerance.
+*Molecular and Cellular Biology* 42(4): e00560-21. https://doi.org/10.1128/mcb.00560
+
+Byrne T, Farrelly N, Kelleher C, et al. (2022).
+Genetic Diversity and Structure of a Diverse Population of Picea sitchensis Using Genotyping-by-Sequencing.
+*Forests* 13(9):1511. https://doi.org/10.3390/f13091511 
+
+Heredia-Pech M, Chávez-Pesqueira M, Ortiz-García MM, t al. (2022). 
+Consequences of introgression and gene flow on the genetic structure and diversity of Lima bean (Phaseolus lunatus L.) in its Mesoamerican diversity area.
+*PeerJ* 10:e13690 https://doi.org/10.7717/peerj.13690
+
+Weisweiler M, Arlt C, Wu PY, et al. (2022).
+Structural variants in the barley gene pool: precision and sensitivity to detect them using short-read sequencing and their association with gene expression and phenotypic variation.
+*Theoretical and Applied Genetics* 135: 3511–3529. https://doi.org/10.1007/s00122-022-04197-7
+
+Liu G, Bracco A, Quattrini AM and Herrera S. (2021).
+Kilometer-Scale Larval Dispersal Processes Predict Metapopulation Connectivity Pathways for Paramuricea biscaya in the Northern Gulf of Mexico.
+*Frontiers in Marine Science* 8:790927. http://doi.org/10.3389/fmars.2021.790927

 Garcia T, Duitama J, Zullo SS, et al. (2021).
 Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.

Aquileo | Home modified by Jorge Duitama

Tue, 10 Jan 2023 23:09:05 -0000

--- v132
+++ v133
@@ -1,34 +1,36 @@
 #NGSEP#
 ###(Next Generation Sequencing Experience Platform)###

-**NGSEP** is an integrated framework for analysis of high throughput sequencing (HTS) reads. The main use of NGSEP is the construction and downstream analysis of large datasets of genomic variation. NGSEP performs accurate detection and genotyping of Single Nucleotide Variants (SNVs), small and large indels, short tandem repeats (STRs), inversions, and Copy Number Variants (CNVs). For version 4 we released a new genomes assembler implementing novel algorithms for the overlap-layout-consensus (OLC) algorithmic approach. As part of this work, we made our own implementation of an FM-index and a minimizers table for alignment of short and long reads. We also released in version 4 a new algorithm for de-novo analysis of Genotype-by-Sequencing reads which supports single-end and paired end GBS and ddRAD-seq data. Finally, we developed a  new graphical interface based on the JavaFX technology. 
-
-**Upgrade from version 3 to version 4**: The command line usage was modified over the entire application to standardize command and option names.  If you wish to upgrade from version 3 to version 4, please take some time to update scripts and follow again our training materials. We apologize for any inconvenience during the upgrade process. We hope that this change will represent an improvement on command line user experience in the long run.
-
-For version 4.0.2 we added support to read CRAM files updating the [hts-jdk](https://github.com/samtools/htsjdk) library. Unfortunately, we found a compatibility issue of this library with java 1.8. For this reason, now we offer full support only for java 11+. Please use the  [discussion forum](https://sourceforge.net/p/ngsep/discussion/) if you have any issue running any functionality of the current version.
+**NGSEP** is an integrated framework for analysis of high throughput sequencing (HTS) reads. The current version of NGSEP includes functionalities for the following main tasks:
+1. Genome assembly from long reads
+2. Library-guided annotation of transposable elements in a genome assembly
+3. Ortholog identification and alignment of annotated genome assemblies
+4. Mapping of long and short reads.
+5. Construction and downstream analysis of large datasets of genomic variation from reads aligned to a reference genome. 
+6. De-novo analysis of Genotype-by-Sequencing reads which supports single-end and paired end GBS and ddRAD-seq data.

 For our latest news, follow us on [Twitter](https://twitter.com/NGSEP): [@NGSEP](https://twitter.com/NGSEP)

 - - - - - -

-Current list of functionalities
+Complete list of functionalities
 ===
 #### De-novo and reference guided reads processing ####

 1.  Read demultiplexing
 2.  Spectrum of k-mer abundances from fastq or fasta files 
 3. Raw reads error correction
-4. **NEW** De-novo assembly of long reads (work still in progress but feel free to try)
+4. De-novo assembly of long reads
 5. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
 6. De-novo analysis of Genotype-by-sequencing (GBS) reads
 7. Alignment of raw reads to a reference genome

 #### Variants discovery and genotyping ####

-1. Integrated analysis of multiple samples for efficient discovery and genotyping of SNVs, indels and STRs. This is now the recommended option for GBS, RAD-sequencingExome sequencing, RNA-seq and low coverage WGS data
+1. Integrated analysis of multiple samples for efficient discovery and genotyping of SNVs, indels and STRs. This is the recommended option for GBS, RAD-sequencingExome sequencing, RNA-seq and low coverage WGS data
 2. Complete individual sample analysis for discovery and genotyping of SNVs, indels, STRs, and CNVs from WGS data
 3. Merging of genotype calls from different samples into a single VCF file
-4. **NEW** Molecular haplotyping of single individuals
+4. Molecular haplotyping of single individuals
 5. Base pair quality and coverage statistics
 6. Distribution of relative allele counts from BAM files
 7. Genome-wide comparison of read depth patterns between two samples
@@ -40,27 +42,29 @@
 2.  Filtering of transcriptome files in GFF3 format
 3.  Large scale alignment of two assembled and annotated genomes.
 4. Identification and clustering of orthologs and paralogs
+5. **NEW**  Library-guided annotation of transposable elements in a genome assembly.
+6. **NEW**  Masking of genome assemblies.

 #### Variants (VCF) downstream analysis ####

-1. Functional annotation of genomic variants
-2. Filtering of VCF files using quality, coverage, and functional criteria
-3. Conversion of VCF files to input formats for several downstream analysis tools such as Mega, Splitstree, Structure, PowerMarker, Flapjack or HapMap
-4. Comparison of genotype calls between VCF files
-5. Calculation of IBS distance matrices from VCF files
-6. Construction of neighbor joining dendograms from distance matrices
-7. Genotype imputation
-8. Calculation of variant density across the genome
-9. Allele sharing statistics for inbred populations
-10. A window-based analysis to discover haplotype introgressions from population VCF files
-11. Alignment of consensus sequences and translation of coordinates for variants obtained with the de-novo analysis of GBS reads
+1. Functional annotation of genomic variants.
+2. Filtering of VCF files using quality, coverage, and functional criteria.
+3. Conversion of VCF files to input formats for several downstream analysis tools such as Splitstree, Structure, PowerMarker, Flapjack or Tassel.
+4. Comparison of genotype calls between VCF files.
+5. Calculation of IBS distance matrices from VCF files.
+6. Neighbor joining clustering from distance matrices.
+7. Genotype imputation.
+8. Calculation of variant density across the genome.
+9. Allele sharing statistics for inbred populations.
+10. A window-based analysis to discover haplotype introgressions from population VCF files.
+11. Alignment of consensus sequences and translation of coordinates for variants obtained with the de-novo analysis of GBS reads.

 #### Benchmark and simulation ####

 1. Simulation of single individuals with different ploidies and variants from a reference genome.
 2.  Simulation of single reads.
-3.  Benchmark statistics comparing test and gold standard VCF files
-4.  Simulation of TILLING experiments
+3.  Benchmark statistics comparing test and gold standard VCF files.
+4.  Simulation of TILLING experiments.

 - - - - - -

@@ -72,7 +76,9 @@
 [Library](http://sourceforge.net/projects/ngsep/files/Library/) to be used in a terminal (Including README for usage instructions).
 [Source Code](https://sourceforge.net/projects/ngsep/files/SourceCode/) bundle for compiling.

-A new graphical interface for NGSEP 4 is now [available](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is [available](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).
+#### Graphical interface : ####
+
+A graphical interface for NGSEP 4 is  [available](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is under construction, but it can be accessed [here](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).

 The Eclipse plugin will no longer be sustained. However, the plugin with the functionalities of NGSEP 3 is still [available](http://sourceforge.net/projects/ngsep/files/OnlyPlugin/). The package including Eclipse and an installer of bowtie2 is also [available](http://sourceforge.net/projects/ngsep/files/Eclipse%2BPlugin/). To update an existing installation, just download the plugin and follow the instructions in the [User Manual](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download).

@@ -97,43 +103,57 @@
 - - - - - - -
 Citing
 ===
+The manuscript of NGSEP 4, focused on orthologs and genome alignment has been recently published at Molecular Ecology Resources:
+
+Tello D, Gonzalez-Garcia LN, Gomez J, et al. (2022).
+NGSEP 4: Efficient and accurate identification of orthogroups and whole-genome alignment.
+*Molecular Ecology Resources in press*. https://doi.org/10.1111/1755-0998.13737
+
+The manuscript with the description of the initial modules of **NGSEP** is available at Nucleic Acids research:
+
+Duitama J, Quintero JC, Cruz DF, Quintero C, Hubmann G, Foulquie-Moreno MR, Verstrepen KJ, Thevelein JM, and Tohme J. (2014). 
+An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments. 
+*Nucleic Acids Research* 42(6): e44.  http://doi.org/10.1093/nar/gkt1381
+
+Details of different algorithms implemented in **NGSEP** can be found in different publications. Feel free to cite the most appropriate paper(s) depending on the analysis task(s) for which **NGSEP** was helpful :
+
+***Variants detection and genotyping:***
+
 The latest algorithms implemented in NGSEP 3 to improve accuracy for variants detection and genotyping can be found at Bioinformatics:

 Tello D, Gil J, Loaiza CD, Riascos JJ, Cardozo N, and Duitama J. (2019)
 NGSEP3: accurate variant calling across species and sequencing protocols.
 *Bioinformatics* 35(22): 4716–4723. http://doi.org/10.1093/bioinformatics/btz275

-Functionalities related to the TILLING experimental setup are described in this recently published manuscript in Frontiers in Genetics:
+***TILLING: ***
+
+Functionalities related to the TILLING experimental setup can be found in Frontiers in Genetics:

 Gil J, Andrade-Martínez JS and Duitama J. (2021)
 Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.
 *Frontiers in Genetics*  12: 54. http://doi.org/10.3389/fgene.2021.624513

+***GBS pipelines: ***
+
 Further details on the pipeline built for reference-guided variants detection on Genotype-By-Sequencing (GBS) data can be found at BMC Genomics:

 Perea C, Hoz JFDL, Cruz DF, Lobaton JD, Izquierdo P, Quintero JC, Raatz B and Duitama J. (2016).
 Bioinformatic analysis of genotype by sequencing (GBS) data with NGSEP.
 *BMC Genomics* 17:498. http://doi.org/10.1186/s12864-016-2827-7

-The manuscript describing the functionality to perform de-novo analysis of GBS reads has been recently published in Molecular Ecology Resources:
+The manuscript describing the functionality to perform de-novo analysis of GBS reads can be found at Molecular Ecology Resources:

 Parra-Salazar A, Gomez J, Lozano-Arce D, Reyes-Herrera PH and Duitama J. (2022).
 Robust and efficient software for reference-free genomic diversity analysis of GBS data on diploid and polyploid species
-Molecular Ecology Resources 22(1): 439-454. http://doi.org/10.1101/2020.11.28.402131
-
-The manuscript with the initial description of the main modules of **NGSEP** is available at Nucleic Acids research:
-
-Duitama J, Quintero JC, Cruz DF, Quintero C, Hubmann G, Foulquie-Moreno MR, Verstrepen KJ, Thevelein JM, and Tohme J. (2014). 
-An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments. 
-*Nucleic Acids Research* 42(6): e44.  http://doi.org/10.1093/nar/gkt1381
-
-Finally, the manuscript describing the genomes aligner is currently in preparation. In the mean time, a great example of use of this functionality is described as part of our recent publication in Nature Communications:
-
-Garcia T, Duitama J, Zullo SS, et al. (2021).
-Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.
-*Nature communications* 12: 702. http://doi.org/10.1038/s41467-021-20921-1
-
-Details of different algorithms implemented in **NGSEP** can be found in different publications. Feel free to cite the most appropriate paper(s) depending on the analysis task(s) for which **NGSEP** was helpful :
+*Molecular Ecology Resources* 22(1): 439-454. http://doi.org/10.1101/2020.11.28.402131
+
+***Genome Assembly: ***
+
+A manuscript describing the algorithms for de-novo genome assembly of long reads is under review. In the mean time, the details of the algorithms are described in this preprint:
+
+Gonzalez-Garcia, L., Guevara-Barrientos D., Lozano-Arce D., *et al*.
+New algorithms for accurate and efficient de-novo genome assembly from long DNA sequencing reads. 
+*bioRxiv* 2022.08.30.505891. https://doi.org/10.1101/2022.08.30.505891 

 ***Molecular haplotyping:***

@@ -169,7 +189,10 @@
 Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection.
 *PLoS ONE* 10(4): e0124617. http://doi.org/10.1371/journal.pone.0124617

-Since version 2.1.2, we implemented a new model to integrate paired-end and split-read analysis for detection of large indels. Benchmark experiments of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:
+
+***Structural variants detection:***
+
+Since version 2.1.2, we implemented a model to integrate paired-end and split-read analysis for detection of large indels. Benchmark experiments of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:

 Fuentes RR, Chebotarov D, Duitama J, Smith S, De la Hoz JF, Mohiyuddin M, et al. (2019).
 Structural variants in 3000 rice genomes.
@@ -186,6 +209,10 @@
 Publications citing NGSEP
 ===
 Different modules of NGSEP have been used to perform bioinformatic analysis or as a baseline for development of novel tools in the following publications:
+
+Garcia T, Duitama J, Zullo SS, et al. (2021).
+Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.
+*Nature communications* 12: 702. http://doi.org/10.1038/s41467-021-20921-1

 Sadohara R, Long Y, Izquierdo P, Urrea CA, Morris D and Cichy K. (2021).
 Seed coat color genetics and genotype × environment effects in yellow beans via machine-learning and genome-wide association.

Aquileo | Home modified by Jorge Duitama

Mon, 10 Jan 2022 23:32:29 -0000

--- v131
+++ v132
@@ -109,17 +109,17 @@
 Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.
 *Frontiers in Genetics*  12: 54. http://doi.org/10.3389/fgene.2021.624513

-Further details on the pipeline built for variants detection on Genotype-By-Sequencing (GBS) data can be found at BMC Genomics:
+Further details on the pipeline built for reference-guided variants detection on Genotype-By-Sequencing (GBS) data can be found at BMC Genomics:

 Perea C, Hoz JFDL, Cruz DF, Lobaton JD, Izquierdo P, Quintero JC, Raatz B and Duitama J. (2016).
 Bioinformatic analysis of genotype by sequencing (GBS) data with NGSEP.
 *BMC Genomics* 17:498. http://doi.org/10.1186/s12864-016-2827-7

-The manuscript describing the recently released functionality to perform de-novo analysis of GBS reads is currently under review. However, the implemented algorithm is described in this preprint:
-
-Parra-Salazar A, Gomez J, Lozano-Arce D, Reyes-Herrera PH and Duitama J. (2020).
+The manuscript describing the functionality to perform de-novo analysis of GBS reads has been recently published in Molecular Ecology Resources:
+
+Parra-Salazar A, Gomez J, Lozano-Arce D, Reyes-Herrera PH and Duitama J. (2022).
 Robust and efficient software for reference-free genomic diversity analysis of GBS data on diploid and polyploid species
-bioRxiv 2020.11.28.402131. http://doi.org/10.1101/2020.11.28.402131
+Molecular Ecology Resources 22(1): 439-454. http://doi.org/10.1101/2020.11.28.402131

 The manuscript with the initial description of the main modules of **NGSEP** is available at Nucleic Acids research:

@@ -187,9 +187,121 @@
 ===
 Different modules of NGSEP have been used to perform bioinformatic analysis or as a baseline for development of novel tools in the following publications:

+Sadohara R, Long Y, Izquierdo P, Urrea CA, Morris D and Cichy K. (2021).
+Seed coat color genetics and genotype × environment effects in yellow beans via machine-learning and genome-wide association.
+*The Plant Genome in press*. https://doi.org/10.1002/tpg2.20173
+
+Galaska MP, Liu G, West D, et al. (2021).
+Seascape Genomics Reveals Metapopulation Connectivity Network of Paramuricea biscaya in the Northern Gulf of Mexico.
+*Frontiers in Marine Science* 8:790929. https://doi.org/10.3389/fmars.2021.790929
+
+Sánchez-Corrales L, Tovar-Aguirre OL, Galeano-Vanegas NF, et al. (2021).
+Phylogenomic analysis and Mycobacterium tuberculosis antibiotic resistance prediction by whole-genome sequencing from clinical isolates of Caldas, Colombia.
+*PLoS ONE* 16(10): e0258402. https://doi.org/10.1371/journal.pone.0258402
+
+Duk M, Kanapin A, Rozhmina T, et al. (2021).
+The Genetic Landscape of Fiber Flax.
+*Frontiers in Plant Science* 12:764612. http://doi.org/10.3389/fpls.2021.764612
+
+Diaz S, Ariza-Suarez D, Ramdeen R, et al. (2021).
+Genetic Architecture and Genomic Prediction of Cooking Time in Common Bean (Phaseolus vulgaris L.).
+*Frontiers in Plant Science* 11:622213. https://doi.org/10.3389/fpls.2020.622213
+
+Soler-Garzón A, Oladzad A, Beaver J, et al. (2021).
+NAC Candidate Gene Marker for bgm-1 and Interaction With QTL for Resistance to Bean Golden Yellow Mosaic Virus in Common Bean.
+*Frontiers in Plant Science* 12:628443. https://doi.org/10.3389/fpls.2021.628443
+
+Garreta L, Cerón-Souza I, Palacio MR, Reyes-Herrera PH. (2021).
+MultiGWAS: An integrative tool for Genome Wide Association Studies in tetraploid organisms.
+*Ecology and Evolution* 11(12): 7411-7426. https://doi.org/10.1002/ece3.7572
+
+Medina C.A., Yu LX. (2021).
+Developing SNPs and Strategies for Genomic Analysis in Alfalfa.
+In: Yu LX., Kole C. (eds) The Alfalfa Genome. Compendium of Plant Genomes. Springer, Cham. https://doi.org/10.1007/978-3-030-74466-3_10
+
+Ayala-Usma DA, Cárdenas M, Guyot R et al. (2021).
+A whole genome duplication drives the genome evolution of Phytophthora betacei, a closely related species to Phytophthora infestans.
+*BMC Genomics* 22: 795. https://doi.org/10.1186/s12864-021-08079-y
+
+Kanapin A, Bankin M, Rozhmina T, Samsonova A, Samsonova M. (2021).
+Genomic Regions Associated with Fusarium Wilt Resistance in Flax.
+*International Journal of Molecular Sciences* 22(22):12383. https://doi.org/10.3390/ijms222212383 
+
+Bello JC, Hausbeck MK and Sakalidis ML. (2021).
+Application of Target Enrichment Sequencing for Population Genetic Analyses of the Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli in Michigan.
+*Molecular Plant Microbe Interaction* 34(10): 1103-1118. https://doi.org/10.1094/MPMI-11-20-0329-TA
+
+Samsonova A, Kanapin A, Bankin M, et al. (2021).
+A Genomic Blueprint of Flax Fungal Parasite Fusarium oxysporum f. sp. lini.
+*International Journal of Molecular Sciences* 22(5):2665. https://doi.org/10.3390/ijms22052665 
+
+Diaz LM, Arredondo V, Ariza-Suarez D, et al. (2021).
+Genetic Analyses and Genomic Predictions of Root Rot Resistance in Common Bean Across Trials and Populations.
+*Frontiers in Plant Science* 12:629221. http://doi.org/10.3389/fpls.2021.629221
+
+Islam M, Abdullah, Zubaida B, et al. (2021).
+Agro-Morphological, Yield, and Genotyping-by-Sequencing Data of Selected Wheat (Triticum aestivum) Germplasm From Pakistan.
+*Frontiers in Genetics* 12: 617772. https://doi.org/10.3389/fgene.2021.617772
+
+García-Fernández C, Campa A, Garzón AS et al. (2021).
+GWAS of pod morphological and color characters in common bean.
+*BMC Plant Biology* 21: 184. https://doi.org/10.1186/s12870-021-02967-x
+
+Negus KL, Chen L, Fresnedo-Ramírez J, Scott HA, Sacks GL, et al. (2021).
+Identification of QTLs for berry acid and tannin in a Vitis aestivalis-derived 'Norton'-based population.
+*Fruit Research* 1: 8. https://doi.org/10.48130/FruRes-2021-0008   
+
+Soler-Garzón A, McClean PE and Miklas PN. (2021).
+Genome-Wide Association Mapping of bc-1 and bc-u Reveals Candidate Genes and New Adjustments to the Host-Pathogen Interaction for Resistance to Bean Common Mosaic Necrosis Virus in Common Bean.
+*Frontiers in Plant Science* 12:699569. http://doi.org/10.3389/fpls.2021.699569
+
+Akhatar J, Goyal A, Kaur N, et al. (2021).
+Genome wide association analyses to understand genetic basis of flowering and plant height under three levels of nitrogen application in Brassica juncea (L.) Czern & Coss.
+*Scientific Reports* 11: 4278. https://doi.org/10.1038/s41598-021-83689-w
+
+Elias JCF, Gonçalves-Vidigal MC, Ariani A, et al. (2021).
+Genome-Environment Association Analysis for Bio-Climatic Variables in Common Bean (Phaseolus vulgaris L.) from Brazil.
+*Plants*. 2021; 10(8):1572. https://doi.org/10.3390/plants10081572 
+
+Grigoreva E, Barbitoff Y, Changalidi A, et al. (2021).
+Development of SNP Set for the Marker-Assisted Selection of Guar (Cyamopsis tetragonoloba (L.) Taub.) Based on a Custom Reference Genome Assembly.
+*Plants* 10(10):2063. https://doi.org/10.3390/plants10102063 
+
+Medina CA, Kaur H, Ray I and Yu LX. (2021).
+Strategies to Increase Prediction Accuracy in Genomic Selection of Complex Traits in Alfalfa (Medicago sativa L.).
+*Cells* 10(12): 3372. https://doi.org/10.3390/cells10123372 
+
+Lobaton J, Andrew R, Duitama J, et al. (2021).
+Using RNA-seq to characterize pollen–stigma interactions for pollination studies.
+*Scientific Reports* 11: 6635. https://doi.org/10.1038/s41598-021-85887-y
+
+Nicolai T, Deparis Q, Foulquié-Moreno MR, et al. (2021).
+In-situ muconic acid extraction reveals sugar consumption bottleneck in a xylose-utilizing Saccharomyces cerevisiae strain.
+*Microbial Cell Factories* 20: 114. https://doi.org/10.1186/s12934-021-01594-3
+
+Miedaner T, Vasquez A, Castiblanco V. et al. (2021).
+Genome-wide association study for deoxynivalenol production and aggressiveness in wheat and rye head blight by resequencing 92 isolates of Fusarium culmorum.
+*BMC Genomics* 22: 630. https://doi.org/10.1186/s12864-021-07931-5
+
+Lin S, Medina CA, Norberg OS, et al. (2021)
+Genome-Wide Association Studies Identifying Multiple Loci Associated With Alfalfa Forage Quality.
+*Frontiers in plant Science* 12:648192. https://doi.org/10.3389/fpls.2021.648192
+
+Deparis Q, Duitama J, Foulquié-Moreno MR, Thevelein JM. (2021).
+Whole-Genome Transformation Promotes tRNA Anticodon Suppressor Mutations under Stress
+*mBio* 12: 2. https://doi.org/10.1128/mBio.03649-20 
+
+Ocampo J, Ovalle T, Labarta R, et al. (2021).
+DNA fingerprinting reveals varietal composition of Vietnamese cassava germplasm (Manihot esculenta Crantz) from farmers’ field and genebank collections.
+*Plant Molecular Biology in press*. https://doi.org/10.1007/s11103-021-01124-0
+
+Musker SD, Ellis AG, Schlebusch SA, Verboom GA. (2021).
+Niche specificity influences gene flow across fine-scale habitat mosaics in Succulent Karoo plants
+*Molecular ecology* 30(1): 175-192. https://doi.org/10.1111/mec.15721
+
 Bassett A, Kamfwa K, Ambachew D and Cichy K. (2021).
 Genetic variability and genome-wide association analysis of flavor and texture in cooked beans (Phaseolus vulgaris L.).
-*Theoretical and Applied Genetics in press* . https://doi.org/10.1007/s00122-020-03745-3
+*Theoretical and Applied Genetics* 134: 959–978. https://doi.org/10.1007/s00122-020-03745-3

 Gupta N, Gupta M, Akhatar J, et al. (2021).
 Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L..

Aquileo | Home modified by Jorge Duitama

Fri, 12 Feb 2021 05:42:47 -0000

--- v130
+++ v131
@@ -18,7 +18,7 @@
 1.  Read demultiplexing
 2.  Spectrum of k-mer abundances from fastq or fasta files 
 3. Raw reads error correction
-4. **NEW** De-novo assembly of long reads
+4. **NEW** De-novo assembly of long reads (work still in progress but feel free to try)
 5. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
 6. De-novo analysis of Genotype-by-sequencing (GBS) reads
 7. Alignment of raw reads to a reference genome
@@ -115,12 +115,24 @@
 Bioinformatic analysis of genotype by sequencing (GBS) data with NGSEP.
 *BMC Genomics* 17:498. http://doi.org/10.1186/s12864-016-2827-7

+The manuscript describing the recently released functionality to perform de-novo analysis of GBS reads is currently under review. However, the implemented algorithm is described in this preprint:
+
+Parra-Salazar A, Gomez J, Lozano-Arce D, Reyes-Herrera PH and Duitama J. (2020).
+Robust and efficient software for reference-free genomic diversity analysis of GBS data on diploid and polyploid species
+bioRxiv 2020.11.28.402131. http://doi.org/10.1101/2020.11.28.402131
+
 The manuscript with the initial description of the main modules of **NGSEP** is available at Nucleic Acids research:

 Duitama J, Quintero JC, Cruz DF, Quintero C, Hubmann G, Foulquie-Moreno MR, Verstrepen KJ, Thevelein JM, and Tohme J. (2014). 
 An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments. 
 *Nucleic Acids Research* 42(6): e44.  http://doi.org/10.1093/nar/gkt1381

+Finally, the manuscript describing the genomes aligner is currently in preparation. In the mean time, a great example of use of this functionality is described as part of our recent publication in Nature Communications:
+
+Garcia T, Duitama J, Zullo SS, et al. (2021).
+Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.
+*Nature communications* 12: 702. http://doi.org/10.1038/s41467-021-20921-1
+
 Details of different algorithms implemented in **NGSEP** can be found in different publications. Feel free to cite the most appropriate paper(s) depending on the analysis task(s) for which **NGSEP** was helpful :

 ***Molecular haplotyping:***
@@ -140,16 +152,19 @@
 *Genome Research* Sep;19(9):1586-1592. http://doi.org/10.1101/gr.092981.109

 ***Genotype imputation:***
+
 Scheet, P and Stephens, M. (2006).
 A Fast and Flexible Statistical Model for Large-Scale Population Genotype Data: Applications to Inferring Missing Genotypes and Haplotypic Phase.
 *American Journal of Human Genetics* 78: 629-644. http://doi.org/10.1086/502802

 ***Read Depth comparison:***
+
 Xie C and Tammi MT. (2009).
 CNV-seq, a new method to detect copy number variation using high-throughput sequencing.
 *BMC Bioinformatics* 10:80.  http://doi.org/10.1186/1471-2105-10-80

 ***Haplotype introgression analysis:***
+
 Duitama J, Silva A, Sanabria Y, Cruz DF, Quintero C, Ballen C, et al. (2015)
 Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection.
 *PLoS ONE* 10(4): e0124617. http://doi.org/10.1371/journal.pone.0124617
@@ -171,10 +186,6 @@
 Publications citing NGSEP
 ===
 Different modules of NGSEP have been used to perform bioinformatic analysis or as a baseline for development of novel tools in the following publications:
-
-Garcia T, Duitama J, Zullo SS, et al. (2021).
-Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.
-*Nature communications* 12: 702. http://doi.org/10.1038/s41467-021-20921-1

 Bassett A, Kamfwa K, Ambachew D and Cichy K. (2021).
 Genetic variability and genome-wide association analysis of flavor and texture in cooked beans (Phaseolus vulgaris L.).

Aquileo | Home modified by Jorge Duitama

Fri, 12 Feb 2021 05:31:42 -0000

--- v129
+++ v130
@@ -1,11 +1,11 @@
 #NGSEP#
 ###(Next Generation Sequencing Experience Platform)###

-**NGSEP** is an integrated framework for analysis of high throughput sequencing (HTS) reads. The main use of NGSEP is the construction and downstream analysis of large datasets of genomic variation. NGSEP performs accurate detection and genotyping of Single Nucleotide Variants (SNVs), small and large indels, short tandem repeats (STRs), inversions, and Copy Number Variants (CNVs). As a roadmap for version 4 we already released our own implementation of an FM-index and a minimizers table for alignment of short and long reads and a new algorithm for de-novo analysis of Genotype-by-Sequencing reads which supports single-end and paired end GBS and ddRAD-seq data. We will also release in version 4 a de-novo genome assembler for long reads (coming soon). Finally, we developed a  new graphical interface based on the JavaFX technology. 
-
-**Upgrade from version 3 to version 4**: The command line usage was modified over the entire application to standardize command and option names.  If you wish to upgrade from version 3 to version 4, please take some time to update scripts and follow again our training materials. We apologize for any inconvinience during the upgrade process. We hope that this change will represent an improvement on command line user experience in the long run.
-
-For version 4.0.2 we added support to read CRAM files updating the [hts-jdk](https://github.com/samtools/htsjdk) library. Unfortunately, we found a compatibility issue of this library with java 1.8. For this reason, now we offer full support only for java 11+. With the exception of the reads aligner, most functionalities still shoud work fine on java 1.8. Please use the  [discussion forum](https://sourceforge.net/p/ngsep/discussion/) if you have any issue running any functionality of this version.
+**NGSEP** is an integrated framework for analysis of high throughput sequencing (HTS) reads. The main use of NGSEP is the construction and downstream analysis of large datasets of genomic variation. NGSEP performs accurate detection and genotyping of Single Nucleotide Variants (SNVs), small and large indels, short tandem repeats (STRs), inversions, and Copy Number Variants (CNVs). For version 4 we released a new genomes assembler implementing novel algorithms for the overlap-layout-consensus (OLC) algorithmic approach. As part of this work, we made our own implementation of an FM-index and a minimizers table for alignment of short and long reads. We also released in version 4 a new algorithm for de-novo analysis of Genotype-by-Sequencing reads which supports single-end and paired end GBS and ddRAD-seq data. Finally, we developed a  new graphical interface based on the JavaFX technology. 
+
+**Upgrade from version 3 to version 4**: The command line usage was modified over the entire application to standardize command and option names.  If you wish to upgrade from version 3 to version 4, please take some time to update scripts and follow again our training materials. We apologize for any inconvenience during the upgrade process. We hope that this change will represent an improvement on command line user experience in the long run.
+
+For version 4.0.2 we added support to read CRAM files updating the [hts-jdk](https://github.com/samtools/htsjdk) library. Unfortunately, we found a compatibility issue of this library with java 1.8. For this reason, now we offer full support only for java 11+. Please use the  [discussion forum](https://sourceforge.net/p/ngsep/discussion/) if you have any issue running any functionality of the current version.

 For our latest news, follow us on [Twitter](https://twitter.com/NGSEP): [@NGSEP](https://twitter.com/NGSEP)

@@ -18,19 +18,21 @@
 1.  Read demultiplexing
 2.  Spectrum of k-mer abundances from fastq or fasta files 
 3. Raw reads error correction
-4. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
-5. De-novo analysis of Genotype-by-sequencing (GBS) reads
-6. Alignment of raw reads to a reference genome
+4. **NEW** De-novo assembly of long reads
+5. Construction of a haploid genome for a sequenced individual from homozygous alternative variants (assembly polishing)
+6. De-novo analysis of Genotype-by-sequencing (GBS) reads
+7. Alignment of raw reads to a reference genome

 #### Variants discovery and genotyping ####

 1. Integrated analysis of multiple samples for efficient discovery and genotyping of SNVs, indels and STRs. This is now the recommended option for GBS, RAD-sequencingExome sequencing, RNA-seq and low coverage WGS data
 2. Complete individual sample analysis for discovery and genotyping of SNVs, indels, STRs, and CNVs from WGS data
 3. Merging of genotype calls from different samples into a single VCF file
-4. Base pair quality and coverage statistics
-5. Distribution of relative allele counts from BAM files
-6. Genome-wide comparison of read depth patterns between two samples
-7. Individual identification (genotyping) for variants identified from pools in TILLING experiments
+4. **NEW** Molecular haplotyping of single individuals
+5. Base pair quality and coverage statistics
+6. Distribution of relative allele counts from BAM files
+7. Genome-wide comparison of read depth patterns between two samples
+8. Individual identification (genotyping) for variants identified from pools in TILLING experiments

 #### Analysis of annotated gene models and transcripts ####

@@ -51,14 +53,14 @@
 8. Calculation of variant density across the genome
 9. Allele sharing statistics for inbred populations
 10. A window-based analysis to discover haplotype introgressions from population VCF files
-11. **NEW**  Alignment of consensus sequences and translation of coordinates for variants obtained with the de-novo analysis of GBS reads
+11. Alignment of consensus sequences and translation of coordinates for variants obtained with the de-novo analysis of GBS reads

 #### Benchmark and simulation ####

 1. Simulation of single individuals with different ploidies and variants from a reference genome.
 2.  Simulation of single reads.
 3.  Benchmark statistics comparing test and gold standard VCF files
-4.  **NEW** Simulation of TILLING experiments
+4.  Simulation of TILLING experiments

 - - - - - -

@@ -70,7 +72,7 @@
 [Library](http://sourceforge.net/projects/ngsep/files/Library/) to be used in a terminal (Including README for usage instructions).
 [Source Code](https://sourceforge.net/projects/ngsep/files/SourceCode/) bundle for compiling.

-A new graphical interface for NGSEP 4 is now [available](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A draft user manual for this interface is [available](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).
+A new graphical interface for NGSEP 4 is now [available](http://sourceforge.net/projects/ngsep/files/GUIFX/). This package has been tested on Linux, MAC and Windows having as only prerrequisite the Java Runtime Environment v11. Unlike the Eclipse plugin, this package does not require any external application. A user manual for this interface is [available](http://sourceforge.net/projects/ngsep/files/training/ManualNGSEP_v4.0.3.pdf).

 The Eclipse plugin will no longer be sustained. However, the plugin with the functionalities of NGSEP 3 is still [available](http://sourceforge.net/projects/ngsep/files/OnlyPlugin/). The package including Eclipse and an installer of bowtie2 is also [available](http://sourceforge.net/projects/ngsep/files/Eclipse%2BPlugin/). To update an existing installation, just download the plugin and follow the instructions in the [User Manual](http://sourceforge.net/projects/ngsep/files/training/UserManualNGSEP_V331.pdf/download).

@@ -78,8 +80,8 @@
 Documentation
 ===
 **Resources for the command line:** 
-1 [Quick Start](https://sourceforge.net/projects/ngsep/files/training/QuickStart.txt/download) to get familiar with the pipeline.
-2 [Tutorial](https://sourceforge.net/projects/ngsep/files/training/Tutorial.txt/download) to learn step-by-step how to use **NGSEP**.
+1 [Quick Start](https://sourceforge.net/projects/ngsep/files/training/QuickStart.txt/download) to get familiar with the pipeline for read alignment and variants detection.
+2 [Tutorial](https://sourceforge.net/projects/ngsep/files/training/Tutorial.txt/download) to learn step-by-step how to use **NGSEP** for read alignment and variants detection.
 3 Bash [Scripts](https://sourceforge.net/projects/ngsep/files/training/) to make things faster.
 4 [README](https://sourceforge.net/projects/ngsep/files/Library/README.txt/download) with a detailed description of all modules and parameters.
 5. See what's new in the [Change Log](https://sourceforge.net/projects/ngsep/files/Library/CHANGELOG/download).
@@ -95,13 +97,18 @@
 - - - - - - -
 Citing
 ===
-
-The latest algorithms implemented in NGSEP 3 to improve accuracy for variants detection and genotyping were recently published in bioinformatics:
+The latest algorithms implemented in NGSEP 3 to improve accuracy for variants detection and genotyping can be found at Bioinformatics:

 Tello D, Gil J, Loaiza CD, Riascos JJ, Cardozo N, and Duitama J. (2019)
 NGSEP3: accurate variant calling across species and sequencing protocols.
 *Bioinformatics* 35(22): 4716–4723. http://doi.org/10.1093/bioinformatics/btz275

+Functionalities related to the TILLING experimental setup are described in this recently published manuscript in Frontiers in Genetics:
+
+Gil J, Andrade-Martínez JS and Duitama J. (2021)
+Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.
+*Frontiers in Genetics*  12: 54. http://doi.org/10.3389/fgene.2021.624513
+
 Further details on the pipeline built for variants detection on Genotype-By-Sequencing (GBS) data can be found at BMC Genomics:

 Perea C, Hoz JFDL, Cruz DF, Lobaton JD, Izquierdo P, Quintero JC, Raatz B and Duitama J. (2016).
@@ -114,7 +121,13 @@
 An integrated framework for discovery and genotyping of genomic variants from high-throughput sequencing experiments. 
 *Nucleic Acids Research* 42(6): e44.  http://doi.org/10.1093/nar/gkt1381

-Details of variant detection algorithms implemented in **NGSEP** can be found in the following publications:
+Details of different algorithms implemented in **NGSEP** can be found in different publications. Feel free to cite the most appropriate paper(s) depending on the analysis task(s) for which **NGSEP** was helpful :
+
+***Molecular haplotyping:***
+
+Duitama J, McEwen GK, Huebsch T, Palczewski S, Schulz S, Verstrepen K, *et al*. (2011)
+Fosmid-based whole genome haplotyping of a HapMap trio child: evaluation of Single Individual Haplotyping techniques.
+*Nucleic Acids Research* 40(5):2041-2053. http://doi.org/10.1093/nar/gkr1042 PMID: 22102577

 ***CNV detection (Read depth analysis):***

@@ -141,7 +154,7 @@
 Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection.
 *PLoS ONE* 10(4): e0124617. http://doi.org/10.1371/journal.pone.0124617

-Since version 2.1.2, we implemented a new model to integrate paired-end and split-read analysis for detection of large indels. A recent benchmark experiment of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:
+Since version 2.1.2, we implemented a new model to integrate paired-end and split-read analysis for detection of large indels. Benchmark experiments of this algorithm against other software tools using data from the 3000 rice genomes project is available at Genome Research:

 Fuentes RR, Chebotarov D, Duitama J, Smith S, De la Hoz JF, Mohiyuddin M, et al. (2019).
 Structural variants in 3000 rice genomes.
@@ -159,9 +172,53 @@
 ===
 Different modules of NGSEP have been used to perform bioinformatic analysis or as a baseline for development of novel tools in the following publications:

+Garcia T, Duitama J, Zullo SS, et al. (2021).
+Comprehensive genomic resources related to domestication and crop improvement traits in Lima bean.
+*Nature communications* 12: 702. http://doi.org/10.1038/s41467-021-20921-1
+
+Bassett A, Kamfwa K, Ambachew D and Cichy K. (2021).
+Genetic variability and genome-wide association analysis of flavor and texture in cooked beans (Phaseolus vulgaris L.).
+*Theoretical and Applied Genetics in press* . https://doi.org/10.1007/s00122-020-03745-3
+
+Gupta N, Gupta M, Akhatar J, et al. (2021).
+Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L..
+*Plant Molecular Biology* 105: 161–175. https://doi.org/10.1007/s11103-020-01076-x
+
+Hill RJ, Baldassi C, Snelling JW, et al.  (2021).
+Fine mapping of the locus controlling self-incompatibility in European hazelnut.
+*Tree Genetics & Genomes *17: 6. http://doi.org/10.1007/s11295-020-01485-5
+
+Bhattarai G, Fennel A, Londo JP, et al. (2021).
+A Novel Grape Downy Mildew Resistance Locus from Vitis rupestris.
+*American Journal of Enology and Viticulture* 72: 12-20. http://doi.org/10.5344/ajev.2020.20030 
+
+Cruz DF, De Meyer S, Ampe J, et al. (2020). 
+Using single‐plant‐omics in the field to link maize genes to functions and phenotypes.
+*Molecular systems biology* 16: e9667. http://doi.org/10.15252/msb.20209667
+
+Musker SD, Ellis AG, Schlebusch SA and Verboom GA. (2020).
+Niche specificity influences gene flow across fine‐scale habitat mosaics in Succulent Karoo plants.
+*Molecular ecology* 30(1) 175-192. http://doi.org/10.1111/mec.15721
+
+Gil J, Herrera M, Duitama J, et al. (2020).
+Genomic Variability of Phytophthora palmivora Isolates from Different Oil Palm Cultivation Regions in Colombia.
+*Phytopathology* 110(9):1553-1564. http://doi.org/10.1094/PHYTO-06-19-0209-R
+
+Valdisser PAMR, Muller BSF, de Almeida Filho JE, et al. (2020).
+Genome-Wide Association Studies Detect Multiple QTLs for Productivity in Mesoamerican Diversity Panel of Common Bean Under Drought Stress.
+*Frontiers in Plant Science* 11: 574674. http://10.3389/fpls.2020.574674
+
+Perez-Fons L, Ovalle TM, Maruthi MN, et al. (2020).
+The metabotyping of an East African cassava diversity panel: A core collection for developing biotic stress tolerance in cassava.
+*PLoS One *15(11): e0242245. http://doi.org/10.1371/journal.pone.0242245
+
+Akhatar J, Singh MP, Sharma A, et al. (2020).
+Association Mapping of Seed Quality Traits Under Varying Conditions of Nitrogen Application in Brassica juncea L. Czern & Coss.
+*Frontiers in Genetics* 11: 744. http://doi.org/10.3389/fgene.2020.00744
+
 Hoyos V, Plaza G, Li X and Caicedo AL. (2020).
 Something old, something new: Evolution of Colombian weedy rice (Oryza spp.) through de novo de‐domestication, exotic gene flow, and hybridization.
-*Evolutionary Applications in press*. http://doi.org/10.1111/eva.12955
+*Evolutionary Applications*  13(8): 1968-1983. http://doi.org/10.1111/eva.12955

 Berry M, Izquierdo P, Jeffery H, et al. (2020).
 QTL analysis of cooking time and quality traits in dry bean (Phaseolus vulgaris L.).
@@ -380,14 +437,6 @@
 Quantitative trait analysis of yeast biodiversity yields novel gene tools for metabolic engineering.
 *Metabolic Engineering* 17:68-81. http://doi.org/10.1016/j.ymben.2013.02.006 PMID: 23518242

-Gaur U, Li K, Mei S and Liu G. (2013)
-Research progress in allele-specific expression and its regulatory mechanisms
-*Journal of Applied Genetics* 54 (3):271–283. http://doi.org/10.1007/s13353-013-0148-y PMID: 23609142
-
-Duitama J, McEwen GK, Huebsch T, Palczewski S, Schulz S, Verstrepen K, *et al*. (2011)
-Fosmid-based whole genome haplotyping of a HapMap trio child: evaluation of Single Individual Haplotyping techniques.
-*Nucleic Acids Research* 40(5):2041-2053. http://doi.org/10.1093/nar/gkr1042 PMID: 22102577
-
 Suk EK, McEwen GK, Duitama J, Nowick K, Schulz S, Palczewski S, *et al*. (2011)
 A comprehensively molecular haplotype-resolved genome of a European individual.
 *Genome research* 21(10):1672-1685. http://doi.org/10.1101/gr.125047.111 PMID: 21813624