Wikiomics:DNA sequencing

Base calling (ABI)

• phred giving more accurate calls for less accurate part of the sequence (like at the end of the run, say 600bp and more) . Phred also gives a probability/quality values for each of the bases allowing more accurate assembly. Quality scores range from 4 to about 60, the "high quality bases" are these with scores > 20. The latest beta version (0.071220.b) supports ABI_3730 as well as older ABI models (373, 377, and 3700), Molecular Dynamics MegaBACE and LI-COR 4000.

1. To run it you need to set up PHRED_PARAMETER_FILE variable.

Bash shell:

export PHRED_PARAMETER_FILE=/path/to/your/file/phredpar.dat

1. To see all the options:

phred -doc | less

1. To do simple basecalling on _all_ files in a input_directory and store the SCF files in scf_output_directory:

phred -id input_directory -cd scf_output_directory:

Caveat: names of the new SCF files will be the same as input files.

• Long Trace & Peak Trace from Nucleics. Claims to increase the lenght of readable bases by ca 80bp. Separate software module for increasing daily throughput of a capillary sequencer. (not tested)
• there has been a number of other papers describing algorithms supposedly superior to phred but working software is not easily obtainable if at all.

Sequence assembly

See and read!: http://www.cbcb.umd.edu/software/

First generation

• phrap
• TIGR assembler
• CAP3 WWW@Pasteur

Genome assemblers used in current genomic projects

• Arachne @Broad Inst (largest number of citations)
• Phusion @Sanger
• Atlas @Baylor

• JAZZ -> @JGI in house only
• RAMEN (not published yet as for 10-02-12), used for medaka and silkworm genome sequencing projects

New Programs

• Minimus suitable for bacterial genomes, part of AMOS
• AMOS A Modular Open-Source Assembler

• EULER P.Pevzner graph algorithm producing superior contigs. Requires phrap and patched ReAligner

• MIRA latest version 2.9.25 enables true hybrid sequence assembly (454 data [GS20 or GS FLX], Solexa with Sanger reads).

• SSAKE program for assembly milions of short sequences

• Newbler Assembler software from 454 for de novo sequence assembly.

Experimental

• SHARCGS, a DNA assembly program designed for de novo assembly of 25-40mer input fragments and deep sequence coverage.

• ALLPATHS (HTML) algorithm only

• Maq mapping short reads to an existing genomic sequence

• SOAP suite of programs, no de novo assembly (yet).

• Bowtie "fast and memory efficient"

The most complete list to date is @seqanswers

See also software from

• GSC Software Centre at Canada's Michael Smith Genome Sciences Centre.

Sequence databases & formats

• SRF a generic format for DNA sequence data
• The Short Read Archive @NCBI

Short reads assembly (Solexa etc)

• Velvet Paper(HTML) De Bruijn Graphs based asembler from EBI (Zerbin & Birney)

Contig ordering/finishing

• Hawkeye interactive visual analytics tool for genome assemblies

Quality control

• TileQC R based program for quality control of Solexa reads