Hi everyone! Does someone want to suggest me some softwares (for Windows) to analyze 454 amplicon library data? What I want to do is analyzing my deep seq data to find variations and I'm looking for a software that helps me in finding real variations (among the background) that are not PCR or sequencing errors. Thank you in advance for your help!!
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Originally posted by Melisa StewartIn the context of a flagship project aiming at understanding and improving the adaptive capacity of ago-ecosystems it will be critical to establish a link between sequence variation, functional variation, gene/protein expression and phenotype adaptation. We will gather NGS data in many genotypes of various species, the genomes of which harbor a high level of structural complexity, with copy number variations, insertions-deletions, whole genome and segmental duplications. These challenging tasks will require bioinformatics developments and implementation of methods for accommodating the high level of repetitiveness of complex genomes. The tools will be integrated into pipelines and made available to end-users through the Galaxy platform. The bioinformatician will therefore also have to provide researchers with advices on their experimental designs in order to ensure compliance of produced datasets with pipelines requirements. The related thread is quite informative and authenticate the significance of the variation in NGS data.
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Just to be more specific, I am looking for a software that gives me the opportunity to adjust cutoffs...in general the software we used for alignment discard the variants below 10% in terms of allelic representation. What I am looking for are variant above and also below 10%
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What type of input/alignments/assemblies/trimming are you working with?
What kind of variants are you looking for SNPs/Indels (I hope it's not indels in homopolymers)?
I would start by reading these recent publications:
Current methods for resolving genetically distinct subclones in tumor samples require somatic mutations to be clustered by allelic frequencies, which are determined by applying a variant calling program to next-generation sequencing data. Such programs were developed to accurately distinguish true p …
Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or …
454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
Here are some possibilities:
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I am working with amplicon generatated by gDNA and run on a 454 GS Junior platform (coverage per amplicon >1000x). I am looking for somatic variants even at low frequencies (missense, splice site mutations, indels not in homopolymers). I am not familiar with bioinformatics that's why I am looking for a software that is simple and can be installed on Windows. Thank you m_two for the publications you suggested
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