Reads That Are Used In De Novo Assemblies That Did Not Map To Contigs

Question

[lwc628]

I did de novo transcriptome assembly using RNAseq reads using an Oases assembler. It has an option to spit out the reads that were unused in the construction of contigs. Using this facility, I divided the raw read (the original fastq file) into used and unused, and used bowtie to map only used reads to the contig. However, mapping was about ~70%, and there were unmapped reads(bowtie has an options to get this)

What can account for the "reads that were used in making of contigs not mapping to the contigs"? Any advice or suggestions are greatly appreciated

[Rm]

See relaxing your bowtie alignment stringency: and building indexes at --offrate 1

Express suggests using bowtie2 with these options : --offrate 1 -a -X 800 --rdg 6,5 --rfg 6,5 --score-min L,-.6,-.4 --no-discordant --no-mixed

Answer 1

[Pavel Senin]

IMO, this might happen due to the error correction which assembly does - i.e. choosing a base for particular position by the majority of aligned reads - then reads with that base being different may not align back (also indels); another issue could be that reads are getting truncated within the assembly process based on the quality, but i am not an expert in Oases... You can investigate this issue further by lowering the alignment stringency when you map your reads back to assembled contigs.

Answer 2

[Jeremy Leipzig]

Any read that spans two contigs won't map to any one contig, although the kmers that make up that read are still "used".

You might ask "why there are multiple contigs at all when there are reads that could connect them?" The answer is that those reads in fact map to multiple contigs because of repetitive regions. Any ambiguities introduced by repeats will force the assembler to keep those contigs separate. So the reads that don't map are likely those that border repeats.