454 Presentation by Teri Mueller

This talk was hosted by Roche. It was a general overview of some of the capabilities of the 454 platform and its bundled software. 454 website - Access to FLX updates and documentation. Register with UCSC account only.

• Pre-talk comment: The GUI is accessible by X11 or VNC.

• 1 bead/1 DNA fragment: filter will try to remove beads with >1 fragment (~20%)
• 200 cycles per FLX Titanium run. New machine does 400.
• 1 flow: single base. Unit of measure for pyrosequencing, as bases get added by flow.
• Amount of light ~ # of bases.
• First 4 bases of each sequence is the key sequence. The first 3 are used to normalize the amount of light for 1 base.
• Library sequences: TCAG standard / GATC rapid. Do not mix them! There are also control sequences (did not catch them)
• 1 mil reads / 2 well plate. Lane masks will decrease number of reads.
• Quality statistics can be viewed in the BaseQualityMetrics and QualityFilterMetrics files.
• 10 hr runs. Processing can take up to 80 hrs running on the default computer. This is mitigated by processing with a cluster.
• 37 gb per run (~28 gb of raw images)

• Amplicon Variant Analyzer: for specific region analysis
• GS Assembler: de Novo or Reference Mapper
• GS Reporter
• GS RunProcessor - Image/signal processing
  • Formats:
  • .cwf: raw image
  • .fna: fasta sequence file, header has a specific format.
  • .qual: quality scores (like Phred, but offset)
  • .sff: standard 454 format. <sfffile -s> will split plate into runs.

• Shotgun vs. Amplicon pipeline defaults
• Keypass (read rejecting):
  • Checks for key sequence
  • ~20% rejection expected
• Dot (read rejecting):
  • Checks for too many negative flows.
  • 3 successive negative flows or N>5% of last positive flow.
• Mix (read rejecting):
  • Checks for too many positive flows.
  • Indication of more than one sequence in bead.
  • >70% positive reads.
• Signal Intensity (read trimming):
  • Reduces size of read until <3% borderline reads.
• Primer (read rejecting):
  • Discard overamplified short sequences.
• Valley (read rejecting):
  • Discard scaled sum scores that are too close to the valleys between base count decision points.
  • Amplicon default: 4/700 0.57%
  • Shotgun default: 4/320 1.25%
• Trim back (read trimming):
  • Like valley, except trims instead of discarding until ratio is acceptable.
  • Amplicon default: off
  • Shotgun default: on
• Quality score trim (read trimming):
  • 40 base window: if error rate >1%, trim a base.
  • <40 bases, throws sequence away.
  • (Even unfiltered, quality scores will reflect low quality areas)
• SFFtools can also perform screen-trimming with a screening db for known contaminants.

A good run: expect a read length mode ~500 and mean >300. ~50% should pass filters.

• All assemblers use .sff files.
• 3, 8 & 20kb paired end libraries.
• Can accept fasta/qual reads.
• 15-25X coverage best.
  • Low coverage: poor contig building
  • High coverage: may cause contig breaks
• 4 bytes per read base in RAM.