Merging two libraries of amplicon

Question

[aswathykgp]

Hi ,

I am Aswathi currently working in V1-V3 amplicon sequencing and analysis. I am doing my sequencing in illumina platform, and as it is not possible to sequence ~515 bp length V1-V3, I did a trial to make two libraries of the V1-V3 amplicon.

One PE library of 2X250 bp covering outer region of the amplicon and second one covering inner region of the same amplicon. Therefore, inner library R1 and R2 reads are overlapping and outer R1 and R2 lies far away. Then I tried to make a overlapped consensus amplicon sequences from these inner R1R2+Outer R1R2 merging together. I have used clustalO for the same. But I am able to produce very less (~5000) amplicon sequences using this methodology.

Can anyone suggest a better tool or approach for making amplicon from these two libraries?

Thank you. Aswathi

Answer 1

[liangjiao.xue]

If you search "paired end merge" you may get some information about software for this task.

FLASh worked well in my case. ( http://ccb.jhu.edu/software/FLASH/ ).

You could also try PEAR:
http://sco.h-its.org/exelixis/web/software/pear/

Well, these two tools only merge paired reads in one library. That means the IDs of each pair is same except /1 or /2 to distinguish the end. These tools may work for inner amplicon, but not for the outer amplicon.
These two tools are NOT for merging to libraries.
To merge two libraries, I would try:
1) Merge inner amplicon with FLASh or PEAR to get Inner_merged set (Set1)
2) Search Set1 with R1 and R2 of outer amplicon, respectively (using blast, blat or other tools)
3) Write one script to get the best hit. R1R2 of outer amplicon, should hit the same fragment of Set1, and create one “faked name” for each pair.
4) Merge R1_outer and Set1 to get Set2
5) Merge Set2 with R2_outter.