Debian Med Next generation sequencing packages

The BEDTools utilities allow one to address common genomics tasks such as finding feature overlaps and computing coverage. The utilities are largely based on four widely-used file formats: BED, GFF/GTF, VCF, and SAM/BAM. Using BEDTools, one can develop sophisticated pipelines that answer complicated research questions by streaming several BEDTools together.

The groupBy utility is distribued in the filo package.

This package addresses the problem to interpret the results from the latest (2010) DNA sequencing technologies. Those will yield fairly short stretches and those cannot be interpreted directly. It is the challenge for tools like Bowtie to give a chromosomal location to the short stretches of DNA sequenced per run.

Bowtie aligns short DNA sequences (reads) to the human genome at a rate of over 25 million 35-bp reads per hour. Bowtie indexes the genome with a Burrows-Wheeler index to keep its memory footprint small: typically about 2.2 GB for the human genome (2.9 GB for paired-end).

Burrows-Wheeler Aligner (BWA) er et program som sammenligner relativt korte nukleotidsekvenser mod lange referencesekvenser, såsom det menneskelige genom. Det implementerer to algoritmer, bwa-short og BWA-SW. Det første virker med forespørgsler som er kortere end 200 bp, og det sidste til længere sekvenser med op til 100 kbp. Begge algoritmer kan sammenligne sekvenser med gab. De er som regel mere akkurate og hurtigere ved forespørgsler med lave fejlrater.

FASTX-Toolkit er en samling af kommandolinjeværktøjer for forbehandling af korte nukleotidlæsninger i FASTA- og FASTQ-formater, normalt udarbejdet af Next-Generation sekvensmaskiner. Hovedbehandlingen af sådanne FASTA/FASTQ-filer er oversættelse (justering) af sekvenserne til referencegenomer eller andre databaser der bruger specialiserede programmer såsom BWA, Bowtie og mange andre. Det er dog undertiden mere produktivt at forbehandle FASTA/FASTQ-filerne, forud for oversættelse af sekvenserne til genom-manipulering, for at fremstille bedre oversættelsesresultater. FASTX-Toolkit-værktøjer udfører nogle af disse forbehandlingsopgaver.

The following tools are available as part of the filo package:

groupBy – mimics the “groupBy” clause in database systems.

shuffle – randomize the order of lines in a file.

stats – computes descriptive statistic on a given column of a tab-delimited file or stream.

Because their name is too generic, ‘shuffle’ and ‘stats’ are relocated in /usr/lib/filo.

LAST er programmel til sammenligning og justering af sekvenser, typisk DNA- eller protein-sekvenser. LAST ligner BLAST, men det er bedre til at håndtere meget store mængder af sekvensdata. Her er to ting som LAST er god til:

• Sammenligning af store (eksempelvis pattedyrs) genomer.
• Kortlægge masser af sekvens-mærker oven på et genom.

Den primære, tekniske innovation er at LAST finder indledende samstemmende forekomster, baseret på deres mangfoldighed, i stedet for at benytte en fast størrelse (BLAST bruger eksempelvis 10-mers). Dette gør, at man kan kortlægge mærker til genomer, uden gentagelsesmaskering (eng. »repeat- masking«), uden at blive overvældet af gentagne forekomster. For at finde disse forekomster som har forskellige størrelser, så bruger den en suffiks- række (inspireret af Vmatch). For at opnå høj følsomhed, så bruger den en usammenhængende suffiks-række, der er en analog til frø som placeres med mellemrum.

Maq (forkortelse af »Mapping and Assembly with Quality«) bygger kortlagte montager fra korte læsninger genereret af næste generation inden for maskiner til sekvensering. Det blev særligt designet til »Illumina-Solexa 1G Genetic Analyzer«, og har indledende funktionalitet til at håndtere ABI SOLid-data. Maq var tidligere kendt som mapass2.

Udvikling af Maq stoppede i 2008. Dets efterfølger er BWA og SAMtools.

Mira er en specialiseret gensekvenssamler for sekvensprojekter regnet som »svære« på grund af et stort antal ensformige gentagelser. For expressed sequence tags-transskriptioner (EST'er) er miraEST specialiseret på rekonstruktion af urørte mRNA-transskriptioner mens det detekterer og klassificerer enlige nukleotidpolymorfismer (SNP), der opstår i forskellige variationer herudfra.

Sekvenssamleren bruges rutinemæssigt til forskellige opgaver såsom mutationsdetektering i forskellige celletyper, analyser af ligheder i transskriptioner mellem organismer og samling af urørte sekvenser fra forskellige kilder for oligodesign i kliniske mikroarrayeksperimenter.

Pakken tilbyder følgende kørbare filer:

• mira: for samling af genomsekvenser
• miramem: estimerer hukommelsen krævet af et projekt. Fungerer ved at lænke til mira.
• convert_project: til konvertering af projektfiltyper til andre typer
• caf2fasta, caf2gbf, caf2text, caf2html, gbf2caf og gbf2fasta er nogle ofte anvendte filkonverteringsprogrammer (fungerer ved at lænke til convert_project)
• scftool: Værktøjssæt som er nyttigt når der arbejdes med SCF-sporfiler
• fastatool: Værktøjssæt som er nyttigt når der arbejdes med FASTA- sporfiler

Indholdte scipts:

• fasta2frag.tcl: fragmentering af sekvenser til mindre, overlappende sekvenser. Nyttig til simulering af haglgeværsekvenser. Kan oprette undersekvenser i begge retninger (/default) og også parrede slutsekvenser.
• fastaselect.tcl: givet en FASTA-fil (og eventuelt en FASTA- kvalitetsfil) og en fil med navne til læsning, vælges sekvenserne fra FASTA (og kvalitetsfil) og skrives til en resulterende FASTA
• fastqselect.tcl: ligesom fastaselect.tcl, men til FASTQ
• fixACE4consed.tcl: Consed indeholder en fejl som gør det umuligt for den at læse konsensusmærker i ACE-filer skrevet af MIRA-samleren (og muligvis andre programmer). Dette skript behandler en ACE-fil sådan at consed kan læse konsensusmærkerne.

Mothur seeks to develop a single piece of open-source, expandable software to fill the bioinformatics needs of the microbial ecology community. It has incorporated the functionality of dotur, sons, treeclimber, s-libshuff, unifrac, and much more. In addition to improving the flexibility of these algorithms, a number of other features including calculators and visualization tools were added.

SAM (Sequence Alignment/Map) format is a generic format for storing large nucleotide sequence alignments. Picard Tools includes these utilities to manipulate SAM and BAM files: BamToBfq IlluminaBasecallsToSam BuildBamIndex MarkDuplicates CalculateHsMetrics MeanQualityByCycle CleanSam MergeBamAlignment CollectAlignmentSummaryMetrics MergeSamFiles CollectGcBiasMetrics NormalizeFasta CollectInsertSizeMetrics QualityScoreDistribution CollectRnaSeqMetrics ReplaceSamHeader CompareSAMs RevertSam CreateSequenceDictionary SamFormatConverter ExtractIlluminaBarcodes SamToFastq EstimateLibraryComplexity SortSam FastqToSam ValidateSamFile FixMateInformation ViewSam

QIIME (canonically pronounced ‘Chime’) is a pipeline for performing microbial community analysis that integrates many third party tools which have become standard in the field. A standard QIIME analysis begins with sequence data from one or more sequencing platforms, including

• Sanger,
• Roche/454, and

• Illumina GAIIx. With all the underlying tools installed, of which not all are yet available in Debian (or any other Linux distribution), QIIME can perform

• library de-multiplexing and quality filtering;

• denoising with PyroNoise;
• OTU and representative set picking with uclust, cdhit, mothur, BLAST, or other tools;
• taxonomy assignment with BLAST or the RDP classifier;
• sequence alignment with PyNAST, muscle, infernal, or other tools;
• phylogeny reconstruction with FastTree, raxml, clearcut, or other tools;
• alpha diversity and rarefaction, including visualization of results, using over 20 metrics including Phylogenetic Diversity, chao1, and observed species;
• beta diversity and rarefaction, including visualization of results, using over 25 metrics including weighted and unweighted UniFrac, Euclidean distance, and Bray-Curtis;
• summarization and visualization of taxonomic composition of samples using pie charts and histograms and many other features.

QIIME includes parallelization capabilities for many of the computationally intensive steps. By default, these are configured to utilize a mutli-core environment, and are easily configured to run in a cluster environment. QIIME is built in Python using the open-source PyCogent toolkit. It makes extensive use of unit tests, and is highly modular to facilitate custom analyses.

Biokonduktor-pakke til differentieret udtryksanalyse af hel transkriptom- sekventering (RNA-seq) og digitale gen-udtryksprofiler med biologisk replikation. Det bruger empirisk Bayes-estimering og præcise test baseret på den negative binomial-fordelingsdistribution. Det er også nyttigt for differentieret signal-analyse med andre typer optællinger af genom-skala.

This tool allows one to display very long data vectors in a space-efficient manner, by organising it along a 2D Hilbert curve. The user can then visually judge the large scale structure and distribution of features simultaenously with the rough shape and intensity of individual features.

In bioinformatics, a typical use case is ChIP-Chip and ChIP-Seq, or basically all the kinds of genomic data, that are conventionally displayed as quantitative track ("wiggle data") in genome browsers such as those provided by Ensembl or UCSC.

Samtools is a set of utilities that manipulate nucleotide sequence alignments in the binary BAM format. It imports from and exports to the ascii SAM (Sequence Alignment/Map) format, does sorting, merging and indexing, and allows to retrieve reads in any regions swiftly. It is designed to work on a stream, and is able to open a BAM (not SAM) file on a remote FTP or HTTP server.

Tools for reading the SRA archive, generally by converting individual runs into some commonly used format such as fastq.

The textual dumpers "sra-dump" and "vdb-dump" are provided in this release as an aid in visual inspection. It is likely that their actual output formatting will be changed in the near future to a stricter, more formalized representation[s]. PLEASE DO NOT RELY UPON THE OUTPUT FORMAT SEEN IN THIS RELEASE.

The "help" information will be improved in near future releases, and the tool options will become standardized across the set. More documentation will also be provided documentation on the NCBI web site.

Tool options may change in the next release. Version 1 tool options will remain supported wherever possible in order to preserve operation of any existing scripts.

The Short Sequence Assembly by K-mer search and 3′ read Extension (SSAKE) is a genomics application for aggressively assembling millions of short nucleotide sequences by progressively searching for perfect 3′-most k-mers using a DNA prefix tree. SSAKE is designed to help leverage the information from short sequences reads by stringently clustering them into contigs that can be used to characterize novel sequencing targets.

Tabix indexes files where some columns indicate sequence coordinates: name (usually a chromosme), start and stop. The input data file must be position sorted and compressed by bgzip (provided in this package), which has a gzip like interface. After indexing, tabix is able to quickly retrieve data lines by chromosomal coordinates. Fast data retrieval also works over network if an URI is given as a file name.

TopHat aligns RNA-Seq reads to mammalian-sized genomes using the ultra high-throughput short read aligner Bowtie, and then analyzes the mapping results to identify splice junctions between exons. TopHat is a collaborative effort between the University of Maryland Center for Bioinformatics and Computational Biology and the University of California, Berkeley Departments of Mathematics and Molecular and Cell Biology.

ECHO is an error correction algorithm designed for short-reads from next-generation sequencing platforms such as Illumina's Genome Analyzer II. The algorithm uses a Bayesian framework to improve the quality of the reads in a given data set by employing maximum a posteriori estimation.

VCFtools is a program package designed for working with VCF files, such as those generated by the 1000 Genomes Project. The aim of VCFtools is to provide methods for working with VCF files: validating, merging, comparing and calculate some basic population genetic statistics.

Velvet er en »de novo«-genomisk assembler som er specielt designet for teknologier til kort læsning, såsom Solexa eller 454, udviklet af Daniel Zerbino og Ewan Birney ved European Bioinformatics Institue (EMBL-EBI), nær Cambridge, i Storbritannien.

Velvet kan i øjeblikket tage korte læsesekvenser, fjerner fejl og fremstiller dernæst unikke »contig'er« i høj kvalitet. Det anvender derefter parvis læseinformation, hvis tilgængelig, til at indhente de gentagne områder mellem contig'erne.

Cufflinks assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples. It accepts aligned RNA-Seq reads and assembles the alignments into a parsimonious set of transcripts. Cufflinks then estimates the relative abundances of these transcripts based on how many reads support each one.

MosaikBuild converts various sequence formats into Mosaik’s native read format. MosaikAligner pairwise aligns each read to a specified series of reference sequences. MosaikSort resolves paired-end reads and sorts the alignments by the reference sequence coordinates. Finally, MosaikText converts alignments to different text-based formats.

At this time, the workflow consists of supplying sequences in FASTA, FASTQ, Illumina Bustard & Gerald, or SRF file formats and producing results in the BLAT axt, the BAM/SAM, the UCSC Genome Browser bed, or the Illumina ELAND formats.

ANNOVAR is an efficient software tool to utilize update-to-date information to functionally annotate genetic variants detected from diverse genomes (including human genome hg18, hg19, as well as mouse, worm, fly, yeast and many others). Given a list of variants with chromosome, start position, end position, reference nucleotide and observed nucleotides, ANNOVAR can perform:

 1. Gene-based annotation: identify whether SNPs or CNVs cause protein coding
    changes and the amino acids that are affected. Users can flexibly use RefSeq
    genes, UCSC genes, ENSEMBL genes, GENCODE genes, or many other gene definition
    systems.
 2. Region-based annotations: identify variants in specific genomic regions,
    for example, conserved regions among 44 species, predicted transcription
    factor binding sites, segmental duplication regions, GWAS hits, database
    of genomic variants, DNAse I hypersensitivity sites, ENCODE
    H3K4Me1/H3K4Me3/H3K27Ac/CTCF sites, ChIP-Seq peaks, RNA-Seq peaks, or many
    other annotations on genomic intervals.
 3. Filter-based annotation: identify variants that are reported in dbSNP,
    or identify the subset of common SNPs (MAF>1%) in the 1000 Genome Project,
    or identify subset of non-synonymous SNPs with SIFT score>0.05, or many
    other annotations on specific mutations.
 4. Other functionalities: Retrieve the nucleotide sequence in any
    user-specific genomic positions in batch, identify a candidate gene list
    for Mendelian diseases from exome data, identify a list of SNPs from
    1000 Genomes that are in strong LD with a GWAS hit, and many other
    creative utilities.

In a modern desktop computer (3GHz Intel Xeon CPU, 8Gb memory), for 4.7 million variants, ANNOVAR requires ~4 minutes to perform gene-based functional annotation, or ~15 minutes to perform stepwise "variants reduction" procedure, making it practical to handle hundreds of human genomes in a day.

Forge Genome Assembler is a parallel, MPI based genome assembler for mixed read types.

Forge is a classic "Overlap layout consensus" genome assembler written by Darren Platt and Dirk Evers. Implemented in C++ and using the parallel MPI library, it runs on one or more machines in a network and can scale to very large numbers of reads provided there is enough collective memory on the machines used. It generates a full consensus alignment of all reads, can handle mixtures of sanger, 454 and illumina reads. There is some support for solid color space and it includes built in tools for vector trimming and contamination screening.

Forge and was originally developed at Exelixis and they have kindly agreed to place the software which underwent much subsequent development outside Exelixis, into the public domain. Forge works with most of the common MPI implementations.