Wikiomics:DNA sequencing

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=Base calling (ABI)=
=Base calling (ABI)=
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* [http://www.phrap.com/phred/ 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.  
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* [http://www.phrap.com/phred/ 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.
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#To run it you need to set up PHRED_PARAMETER_FILE variable.  
#To run it you need to set up PHRED_PARAMETER_FILE variable.  
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* [http://www.nucleics.com/ 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)
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* [http://www.genome.org/cgi/content/full/11/5/875 LifeTrace] from Incyte. Usage info from [https://www.cebitec.uni-bielefeld.de/cgi-bin/man.cgi?section=1&topic=lifetrace U.Bielefeld]. Comparable with phred, better than phred on MegaBACE sequences. Not available from original Incyte web site (080624).
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* there has been a number of other papers describing algorithms supposedly superior to phred but working software is not easily obtainable if at all.
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* [http://www.nucleics.com/ 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.
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=Sequence assembly=
=Sequence assembly=

Revision as of 17:30, 12 February 2010

Contents

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

Genome assemblers used in current genomic projects

  • JAZZ -> @JGI in house only
  • RAMEN (not published yet as for 08-6-17), 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

Experimental

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

Sequence databases & formats

Short reads assembly (Solexa etc)

Contig ordering/finishing

  • Hawkeye interactive visual analytics tool for genome assemblies

Quality control

  • TileQC R based program for quality control of Solexa reads
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