Paired-End Sequencing from MiSeq

[SDPA_Pet]

Hi guys,

I am a newbie for Illumina sequencing. I just got my sequencing results back from a sequencing center. I am doing environmental microbial amplicon sequencing (16S rRNA). The center uses paired-end. Here is what I think. When they do paried-end sequencing. They sequences twice. First, use forward primer to sequence (5'--3'). Second, use reverse primer to sequence from other direction (3'-5'). For each sample, I have to sequence results and I can assemble them to a large sequences (contig, whatever it called).

Here are files that I got from the sequencing center

1> They give me several files: fasta, qual, fastaq files. These three files are same origin. I was told they are RAW data that have been joined (for amplicons > 300 and < 570bp). Also, I was told that they are joined reads as well as reorienting the 3’-5’ reads into a uniform direction.

Here is my first question: When I check the joined reads. I only find forward primers and barcodes, but there are no reverse primers and barcodes. Does this mean they have removed the reversed primers+barcodes. I suspect they only did a single direction sequencing. My reads is kind of short. The median is ~ 150bp after remove primers and barcodes

2> They also gave me two text files. According to them, if you amplicons are short (<300bp), they don't join them. Theoretically, my amplicons are longer than 300bp, based on the length between the forward and reverse primers. I don't know why they still give me these two files.


Inside the text file, it looks somthing like this

M02542:80:000000000-AFKAB:1:1116:23915:4775
M02542:80:000000000-AFKAB:1:1115:7131:7895

Does illumina sequencing generate txt file? I just switch from 454 to illumina sequencing. Text file in 454 sequencing is raw flowgram file. What does text file in illlumina mean. I haven't heard illumina generates txt file before.

3>Last, I need to redesign some of my old 454 primers for Miseq sequencing. Do you know the maximum length of amplicon I can make for paired-ends sequencing in Miseq

[fanli]

When I check the joined reads. I only find forward primers and barcodes, but there are no reverse primers and barcodes. Does this mean they have removed the reversed primers+barcodes. I suspect they only did a single direction sequencing. My reads is kind of short. The median is ~ 150bp after remove primers and barcodes

Do you mean you have a forward barcode? Because generally you should have this in order to demultiplex.

Theoretically, my amplicons are longer than 300bp, based on the length between the forward and reverse primers.

So you really don't want the contigs that are <300bp then..

Does illumina sequencing generate txt file?

Your typical output is a FASTQ file.

A bit more information would be helpful. For example, what amplicon construct did you use? What sequencing chemistry did you use (e.g. 2x150, 2x250)?

[SDPA_Pet]

Hi Fanli,

Thanks,

The first, yes I can demulitplex. I am just curious where is reversed primers or barcodes. Do they normally remove it for you after joined?


the last question. I have no idea what chemistry for current center use. I will try another center. They will use 2*250. Does this mean I can maximum amplify 500bp? for 2*150 maxmum 300bp? This include primers+barcodes or not?

[microgirl123]

Generally, with amplicon sequencing you want a good bit of overlap (some people favor a complete overlap) between your forward and reverse reads. So a 500-cycle (2x250 bp) kit would be appropriate for something like a 400 bp amplicon so that you have a 100 bp overlap. This overlap allows whatever bioinformatics software you are using to join your forward and reverse reads back into a single amplicon even if the quality of your reads has decreased toward the ends (which is common with Illumina sequencing).

Unless you have added inline barcodes (inside the Illumina adapter/barcodes) when creating your library, you should not be seeing the barcodes. The MiSeq (which is what I think you are using?) should be demultiplexing your samples for you. The index reads on a MiSeq are completely separate from the sequencing reads, and the data does not show up in your fastq files.

The only way to see the Illumina barcodes in your reads is if you sequence through your amplicon into the adapter/barcode on the other side. With a 2x250 read this would happen if your amplicon was less than 200 bp (or if you were sequencing adapter dimers that have no insert).