<p>I have illumina paired-end whole-genome sequencing reads which I have map to around -400 reference plastid genomes. After getting mapped reads, I have to assemble as de novo plastid genome.</p>

<p>1. Do I have map reads to invidiual reference genomes one by one, or can I download all genomes at one go and index as one reference genome?. Do bwa or bowtie has<br />
enough memory to index 400 genomes as one reference index genome?</p>

<p>2. Do you think which one is best method?. Mapping individual genome or all genomes indexed as one?</p>

<p>3. If I have to map individually, can I combine all bam file together and Can I convert to fastq file using bam2fastq tool (in picard) for denovo assembly?</p>

<s>I don't fully understand your introductory sentence as stated, but I can answer your questions to the best of my understanding:</s>

1. You can concatenate all of your genomes into one large file and then index that composite genome. Just make sure your naming convention for each component in the FASTA is logical so that you can understand your results downstream. bwa and bowtie don't "have their own memory," but if you're using a 64-bit system you shouldn't run into any size limitation troubles, especially with plastid sequences.

2. **EDIT:** I say map to a single composite reference. You can pass parameters into bowtie to limit your mappings on the front end, thus saving computational time and making it easier to isolate the most accurate mappings for each read.

3. You can merge BAM files using <a href="http://samtools.sourceforge.net/samtools.shtml">samtools merge</a>, but of course you wouldn't need to if you proceed according to my recommendation. There are a number of tools from converting from BAM back to FASTQ, and the Picard tool should work just fine. It does have trouble with paired-end mappings in certain circumstances though, and if you run into trouble using Picard I'd suggest <a href="http://bedtools.readthedocs.org/en/latest/content/tools/bamtofastq.html">bedtools bamtofastq</a>.

<s>Is the idea here that you're going to map to a bunch of plastid reference sequences from various organisms, and then convert the aggregate mappings back to FASTQ and perform an assembly from them? If so, I say concatenate the reference sequences into one file and map against that. That way it will be easier to retain the best mappings up front, especially if you don't care about *which* reference you're mapping to.</s>