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Hello -
I am trying to assemble the chloroplast genome of an un-culturable diatom. This is the first time I have tried to assemble and finish something like this, and I have no prior experience, so please bear with me. The data I am working with for this project includes ~11 million reads I have generated over four separate genomic shotgun Ion Torrent PGM runs. None of the libraries used paired end sequencing.
From an initial de novo assembly in CLC Genomics Workbench, I have identified three contigs of chloroplast DNA (~13kbp, ~50kbp, ~47kbp respectively, all with ~400X coverage) and that form a nearly complete chloroplast genome. More specifically, through BLAST I have oriented/ordered these contigs against the published Phaeodactylum tricornutum chloroplast genome. The total length of the P. tricornutum chloroplast genome is ~117kbp, and my three contigs add up to ~111kbps. Two of the gaps in my contigs are likely very small (maybe a couple hundred base pairs based on how they match up with Phaeodactylum), whereas the largest gap corresponds almost exactly with Inverted Repeat A. One of the inverted repeats appears to be completely assembled in one of my contigs (IRb), whereas IRa is not. While I can easily design primers to bridge the small gaps, I have no idea what to do about the inverted repeat.
I understand (I think) how the de novo assembly would not make two copies of the inverted repeat, so I was expecting this. I am assuming that the diatom I am working with has IRa and IRb, but how to I determine this? How do I resolve this missing piece?
I have also tried mapping all of the raw reads to each of eight different diatom chloroplast genomes, pooling the reads, removing duplicates, and then mapping to Phaeodactylum. With this approach, I get high mapping coverage of both IRa and IRb.
Since the genes on either side of each repeat are different, do I take the approach of sequencing all the four IR junctions with the rest of the chloroplast to prove IRa is there? That still doesn't tell me the full sequence of IRa - I assume it is identical to IRb, but how can I be sure? I apologize if these are stupid questions, but I have not found much useful information for this specific issue among the papers I have found in the literature so far that deal with assembly of chloroplast genomes from NGS data.
Any help or advice on how to proceed is welcomed. Thanks -
I am trying to assemble the chloroplast genome of an un-culturable diatom. This is the first time I have tried to assemble and finish something like this, and I have no prior experience, so please bear with me. The data I am working with for this project includes ~11 million reads I have generated over four separate genomic shotgun Ion Torrent PGM runs. None of the libraries used paired end sequencing.
From an initial de novo assembly in CLC Genomics Workbench, I have identified three contigs of chloroplast DNA (~13kbp, ~50kbp, ~47kbp respectively, all with ~400X coverage) and that form a nearly complete chloroplast genome. More specifically, through BLAST I have oriented/ordered these contigs against the published Phaeodactylum tricornutum chloroplast genome. The total length of the P. tricornutum chloroplast genome is ~117kbp, and my three contigs add up to ~111kbps. Two of the gaps in my contigs are likely very small (maybe a couple hundred base pairs based on how they match up with Phaeodactylum), whereas the largest gap corresponds almost exactly with Inverted Repeat A. One of the inverted repeats appears to be completely assembled in one of my contigs (IRb), whereas IRa is not. While I can easily design primers to bridge the small gaps, I have no idea what to do about the inverted repeat.
I understand (I think) how the de novo assembly would not make two copies of the inverted repeat, so I was expecting this. I am assuming that the diatom I am working with has IRa and IRb, but how to I determine this? How do I resolve this missing piece?
I have also tried mapping all of the raw reads to each of eight different diatom chloroplast genomes, pooling the reads, removing duplicates, and then mapping to Phaeodactylum. With this approach, I get high mapping coverage of both IRa and IRb.
Since the genes on either side of each repeat are different, do I take the approach of sequencing all the four IR junctions with the rest of the chloroplast to prove IRa is there? That still doesn't tell me the full sequence of IRa - I assume it is identical to IRb, but how can I be sure? I apologize if these are stupid questions, but I have not found much useful information for this specific issue among the papers I have found in the literature so far that deal with assembly of chloroplast genomes from NGS data.
Any help or advice on how to proceed is welcomed. Thanks -
I have the same question. Have you solved it/?
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