I'm planning to sequence complete chloroplast genomes. In some cases I don't have reference genomes from the same genus, although, I have plastomes already sequenced from the same plant family or subfamily. My question is: can I use short 100 bp Illumina pared-end reads to assemble the genome using the family level relatives as references? Usually such short reads (100bp PE) are very problematic when used in the absence of the reference genome. Or I have to use longer 300 bp illumina reads for that?
Dear Peter Poczai,
You can use chloroplast genome sequence of a specie in the same family as a reference genome. Sometimes, I use reference sequences from different families in the same order. There was no problem for assembly. If you find some gaps you can use assembled sequences as the new reference to do the second assembly.
Of course, 300 bp reads are better than 100 bp reads. However, we can use 100 bp reads to assemble the genome.
Best regards,
Khoa
We have learned from experience that the amount of chloroplast sequences you get is so high that you are able to assemble complete chloroplast genomes using a reference that is close enough to your favorite species. Even for 100bp reads.
Thanks for the answers. I made some alignments to compare the available plastomes of related species and the differences are not so huge. I could even use the references of the same order. My samples are all eudycots.
Hi, I developed a new de novo assembler for plastids and it should assemble the chloroplast genome in one circular contig, at least if there is enough data.. I tested it on different datasets of 101 bp illumina reads and it was always successful, you need one chloroplast read, a short sequence or a distant chloroplast genome as a seed. I will upload the assembler in the next few weeks if you would be interested: https://github.com/ndierckx/NOVOPlasty