PacBio vs MinION

BioKiwi · 06-19-2015, 02:16 AM

To compare PacBio long reads with MinION for de novo assembly of bacterial genomes what one should consider for library prep and assembly.

luc · 06-19-2015, 09:35 AM

I am not sure what you are asking for. The PacBio option works: 1 library prep, sequence 1 SMRT-cell & assemble the data - you get a "finished" genome. If playing around with new technology is your goal, then the minion is worth considering.

nucacidhunter · 06-19-2015, 05:45 PM

If the aim is to compare performance of two platforms. I would use the same sheared and size selected DNA from a bacteria with reference genome to prepare libraries for each platform. Then would sequence them to a certain Mb or Gb (can subsample reads to equal bases if one platform output is higher than the other) and use them for assembly and alignment. Metrics such as N50 (L50), contig number, proportion of data used for assembly, evenness of coverage, gaps and GC biases can be used for comparison.

BioKiwi · 06-21-2015, 01:44 AM

Thanks for replies. I wonder if I can use similar approach for transcript isoform analysis.

Brian Bushnell · 06-21-2015, 09:33 AM

With mapping and assembly of DNA, to a known reference, it is relatively straightforward to determine which platform is better.

With isoform analysis, you're bringing in a huge number of additional variables; you won't know the correct answer, so you will have very little ability to evaluate which platform is doing a better job.

nucacidhunter · 06-22-2015, 02:44 AM

I agree with Brian about isoform analysis potential issues. With current knowledge the best option is to access a well annotated fungal species with small genome or C. elegans (to keep sequencing cost down) and prepare full-length dscDNA from mRNA fraction using SMART technology and prepare libraries for both platforms. Transcripts sequences can be used for comparison against annotated regions and genome to compare the coverage of known isoforms and potential new isoform discovery of two platforms.

maxsalm · 06-22-2015, 03:10 AM

Another option may be to get in touch with the Snyder lab and study the same transcriptomes used in their exploration of PacBio long-reads:

http://www.nature.com/nbt/journal/va.../nbt.3242.html
http://www.nature.com/nbt/journal/v3.../nbt.2705.html
http://www.pnas.org/content/111/27/9869.abstract

BioKiwi · 06-23-2015, 02:48 AM

Thank you very much everyone for your posts. I have much clearer idea to plan my Honours project.

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06-19-2015, 02:16 AM	#1
BioKiwi Member Location: KiwiTeritory Join Date: Sep 2014 Posts: 19	PacBio vs MinION To compare PacBio long reads with MinION for de novo assembly of bacterial genomes what one should consider for library prep and assembly.

06-19-2015, 05:45 PM	#3
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	If the aim is to compare performance of two platforms. I would use the same sheared and size selected DNA from a bacteria with reference genome to prepare libraries for each platform. Then would sequence them to a certain Mb or Gb (can subsample reads to equal bases if one platform output is higher than the other) and use them for assembly and alignment. Metrics such as N50 (L50), contig number, proportion of data used for assembly, evenness of coverage, gaps and GC biases can be used for comparison. Last edited by nucacidhunter; 06-19-2015 at 07:34 PM.

06-19-2015, 09:35 AM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	I am not sure what you are asking for. The PacBio option works: 1 library prep, sequence 1 SMRT-cell & assemble the data - you get a "finished" genome. If playing around with new technology is your goal, then the minion is worth considering.

06-21-2015, 01:44 AM	#4
BioKiwi Member Location: KiwiTeritory Join Date: Sep 2014 Posts: 19	Thanks for replies. I wonder if I can use similar approach for transcript isoform analysis.

06-21-2015, 09:33 AM	#5
Brian Bushnell Super Moderator Location: Walnut Creek, CA Join Date: Jan 2014 Posts: 2,707	With mapping and assembly of DNA, to a known reference, it is relatively straightforward to determine which platform is better. With isoform analysis, you're bringing in a huge number of additional variables; you won't know the correct answer, so you will have very little ability to evaluate which platform is doing a better job.

06-22-2015, 02:44 AM	#6
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	I agree with Brian about isoform analysis potential issues. With current knowledge the best option is to access a well annotated fungal species with small genome or C. elegans (to keep sequencing cost down) and prepare full-length dscDNA from mRNA fraction using SMART technology and prepare libraries for both platforms. Transcripts sequences can be used for comparison against annotated regions and genome to compare the coverage of known isoforms and potential new isoform discovery of two platforms.

06-22-2015, 03:10 AM	#7
maxsalm Member Location: London Join Date: Feb 2015 Posts: 18	Another option may be to get in touch with the Snyder lab and study the same transcriptomes used in their exploration of PacBio long-reads: http://www.nature.com/nbt/journal/va.../nbt.3242.html http://www.nature.com/nbt/journal/v3.../nbt.2705.html http://www.pnas.org/content/111/27/9869.abstract

06-23-2015, 02:48 AM	#8
BioKiwi Member Location: KiwiTeritory Join Date: Sep 2014 Posts: 19	Thank you very much everyone for your posts. I have much clearer idea to plan my Honours project.