How to sequence a bacterial genome at the end of 2012

A potential user (‘customer’) of our sequencing platform asked how to generate reference genomes for his 4 bacterial strains. His question inspired me to write this post. The suggestions below are not absolute, just my thoughts on how one these days could go about sequencing a bacterial genome using one or more of the sequencing platforms. I would appreciate any feedback/suggestions in the comments section!

Option 1: bits and pieces

• Libraries: paired end or single end sequencing
• Platform: one or more of Illumina MiSeq or HiSeq, Ion Torrent PGM, 454 GS FLX or GS Junior
• Bioinformatics: assembly: Velvet, SOAPdenovo, Newbler, MIRA, Celera
• Outcome: up to hundreds of short contigs (with only single-end reads) or contigs + scaffolds (with paired end reads)
• Pros: fast and cheap, OK for presence/absence of e.g. genes
• Cons: doesn’t give much insight into the genome
• Remarks: due to per-run throughput, multiplexing is recommended; data can also be used for mapping against a reference genome instead

Option 2: a few gaps remaining

• Libraries: paired end or single end sequencing combined with at least one mate pair library of 8kb distance, optionally a 3-5 kb mate pair library
• Platform: paired end: one or more of Illumina MiSeq or HiSeq, Ion Torrent PGM, 454 GS FLX or GS Junior; mate pairs: preferably on the 454, otherwise Illumina, demonstrated protocol for Ion PGM
• Bioinformatics: assembly: Illumina data: Velvet, SOAPdenovo; 454 data: Newbler MIRA, Celera; hybrid: Newbler, Celera
• Outcome: down to one or a few scaffolds per chromosome/plasmid with a few to numerous gaps remaining
• Pros: gives a lot of long-range information
• Cons: mate pair libraries more cumbersome to make; more expensive, remaining gaps often due to repeats, which may be exactly the regions of interest (e.g. transposons, rDNA operons)
• Remarks: hybrid assembly is largely uncharted territory; gap closing programs may help (IMAGE2, Soap gap closer, GapFiller, …)

Option 3: closing the gaps

• Libraries: as for option 2, but in addition a long-insert (10kb) PacBio library
• Platform: one of Illumina MiSeq or HiSeq, Ion Torrent PGM, 454 GS FLX or GS Junior; plus PacBio RS
• Bioinformatics: assembly: same as option 2; gap closing/finishing: PBJelly; AHA from PacBio can also be used
• Outcome: down to one or a few scaffolds per chromosome/plasmid potentially without gaps remaining
• Pros: no mate pairs needed, potentially very complete genome
• Cons: requires multiple libraries, expensive
• Remarks: Quiver can be used to improve the per-base quality given enough PacBio coverage

Option 4: The ALLPATHS_LG way

• Libraries: Illumina paired end + Illumina Mate Pair + a long-insert (10kb) PacBio library
• Platform: Illumina HiSeq or MiSeq; PacBio RS
• Bioinformatics: assembly: ALLPATHS_LG
• Outcome: down to one or a few contigs per chromosome/plasmid
• Pros: can yield finished genome
• Cons: complex mixture of libraries, can become expensive
• Remarks: special recipe: the paired end library should have a short insert, such that the forward and reverse read overlap

Option 5: PacBio hybrid

• Libraries: paired end or single end sequencing + a long-insert (10kb) PacBio library
• Platform: as for option 2, but PacBio RS in addition
• Bioinformatics: error correction of the PacBio reads: PacBioToCA, LSC, P_ErrorCorrection from PacBio; assembly: Celera, MIRA
• Outcome: down to one or a few scaffolds per chromosome/plasmid potentially without gaps remaining
• Pros: can yield finished genome; only needs two libraries that are simple to produce
• Cons: error-correction can be computationally demanding
• Remarks: Quiver can be used to improve the per-base quality given enough PacBio coverage

Option 6: PacBio only

• Libraries: a single, long-insert (10kb) PacBio library
• Platform: PacBio RS
• Bioinformatics: error-correction; HGAp; assembly: Celera or Allora
• Outcome: down to one or a very few contigs per chromosome/plasmid
• Pros: uses a single library, cheap, finished genome of very high quality
• Cons: somewhat experimental, limited examples available, therefore currently a bit more risky
• Remarks: use Quiver for polishing

EDIT: additional tools

• Reader BenK suggested adding optical mapping as a way to improve bacterial genome assemblies. Not a sequencing technology per se, but, as he writes, “Many genomes have undiscovered repetitive regions that mapping unveils.”
• Reader Roy suggested mentioning stand-alone scaffolders: SSPACE, SOPRA, and the reference-based scaffolder ABACUS . See also this SeqWiki howto.
• Sébastien Boisvert, author of Ray, suggested I add his program. Done hereby. The reason that it is not listed above has to do with my inexperience with it with regards to de novo bacterial genome assemblies
• In the same spirit, reads may want to look at the GAGE results for more programs.

Relevant previous blog posts:

Combining short and long reads: choosing between PacBioToCA and the new ALLPATHS_LG

Ion Torrent Mate Pairs and a single scaffold for E coli K12 substr. MG1655

Links to programs mentioned:

ALLPATHS_LG
Celera
GapFiller
HGAp
IMAGE2
LSC
MIRA
Newbler: request here
P_ErrorCorrection, Allora and AHA see here, these need smrtpipe
PacBioToCA (also part of Celera 7.0 and smrtpipe)
PBJelly
Quiver see here
SOAPdenovo 
Velvet

Reader Roy suggested adding Spades.

(If you feel I missed a program here, let me know in the comments below!)

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