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Chambwe bosc2010
- 2. Applications of Next Generation Sequencing McPherson J.D. Nat Methods. 2009
- 3. Next Generation Sequencers Metzker, M.L. Nat Rev Genet. 2010 Roche/454 GS FLX Titanium Illumina/Solexa GA IIe Life Technologies SOLiD 3 Helicos BioSciences Heliscope NGS Chemistry Pyrosequencing Reversible Terminators Sequencing by ligation Reversible Terminators Avg Read Length (bp) 330 75 50 32 Run Time (days) 0.35 4 7 8 Giga bases/run 0.45 18 30 37 Million reads/run 1.36 240 600 1156
- 6. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python RNA-Seq Pipeline IGV Plug-in The Goby Software Framework reads alignments histograms Readers Writers Iterators File Format Conversions Alignment Processing Visualization
- 7. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python RNA-Seq Pipeline IGV Plug-in The Goby Software Framework Readers Writers Iterators File Format Conversions Alignment Processing Visualization
- 13. Goby File Size Comparisons MAQC sample B = Ambion Human Brain Reference RNA (HBRR or HBR, Catalog #6050) sequenced on four next-gen platforms
- 14. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python Readers Writers Iterators The Goby Software Framework reads alignments histograms File Format Conversions Alignment Processing Visualization RNA-Seq Pipeline IGV Plug-in
- 15. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python Readers Writers Iterators RNA-Seq Pipeline IGV Plug-in The Goby Software Framework reads alignments histograms File Format Conversions Alignment Processing Visualization
- 17. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python RNA-Seq Pipeline IGV Plug-in The Goby Software Framework reads alignments histograms Readers Writers Iterators File Format Conversions Alignment Processing Visualization
- 18. File Formats Low Level APIs Tools/Utilities Applications Java, C++, Python RNA-Seq Pipeline IGV Plug-in The Goby Software Framework reads alignments histograms Readers Writers Iterators File Format Conversions Alignment Processing Visualization
- 24. cDNA Search
Hinweis der Redaktion
- Applications of NGS include Explosion of NGS A gap exists between current sequence-generation and data analysis capabilities to extract relevant biological insights
- Several sequencing platforms available on the market Each with unique chemistry and producing data with different characteristics Throughput varies very large
- Preponderance of NGS data file formats to represent these data
- Here is a list of characteristics we find desirable in a NGS file format Transition: Developed file formats that meet these requirements. File formats are not sufficient therefore we have developed a framework to use these formats and create analysis tools
- This is an outline of the Goby Software Framework
- Now I will discuss File formats
- PB think xml but better
- Brief overview of how schemas are written using PBs A collection of messages of type readEntries
- Transition: to achieve compression we gzip collections of messages
- Protocol buffers do not support messages larger than a few megabytes Contribution of Goby is implementing Protocol buffers in such way to remove the collection size limitation scale for very large messages Overcome by splitting messages into chunks Each Chunk of a compact reads file represents 10,000 or less ReadEntry messages Supports semi random access Chunking leveraged for parallel processing – different servers can access chunks independently - Semi Random Access
- Gzip and chunking meet the requirements for random access and streaming Transition: how well do we do with respect to file sizes
- Apple --- apples comparison Multiple alignments
- Formats are compact How can YOU use it? Low level API’s
- One practical example of printing entries in an alignment file Goby makes it easy to write code to iterate over the contents of multiple compact alignment files
- Goby provides utilities to help build analysis pipeline
- MAQC sample B = Ambion Human Brain Reference RNA (HBRR or HBR, Catalog #6050) sequenced on multiple platforms Normalized gene expression counts RPKM Random hexamer priming results in a bias in nucleotide composition at the start of sequence reads Hansen KD. et al. Nucleic Acids Res. 2010 Jul 1;38(12):e131. Epub2010 Apr 14 Hansen Reweighting scheme to correct for that bias implemented in Goby for genes
- Ambion Human Brain Reference RNA -- MAQCII sample B Different Brain regions from 23 donors.