pyrotag process

[plumb]

I am new to pyrosequencing. We got some 16s rRNA data from sequencing center. It's fasta format file. they give me two set of the data:

prepared 2 sets of files (zips attached);
1) reads for each amplicon starting from the FLXA side (> 200 bp)
2) reads for each amplicon starting from the FLXB side (> 200 bp)

What's does that mean?

We are using that to estimate the biodiversity in the sample. which data set I should use?


They didn't give me quality file either. Is the quality file very important in the quality control step of the data process?

[krobison]

I don't have experience with this sort of data, but there is a general approach you can take to puzzle this out and which will help you understand what you have.

If you have two files of sequences from a sequencing experiment, there are three probable relationships between them

A) They are uncorrelated
B) They are both derived from the same input sequence with an unread spacer separating them and in opposite orientation
C) They are both derived from the same input sequence with an unread spacer separating them, but in the same orientation

First, take about 20 reads from the top of each file (easy to write a program in your favorite language) and put them in new files.

Okay, are the identifiers in the two files related to each other in a pairwise fashion? If so, then either B or C is likely.

Next, use BLASTN to identify best matches for all of these in a database such as nr. Now look at the alignments. Are there pairs of sequences aligning to the same hit? In such pairs, are the hits in the same orientation or opposite orientation?

Even if you did think you knew what the files SHOULD contain, these steps are a useful QC step.

[plumb]

figured that out. one set of file is from the forward reading and another is from reverse reading. I need to combine these sequence properly. Otherwise, we may double count the organism.

Any suggestion for that?

[kmcarr]

Plumb,

In 454 Amplicon sequencing, the PCR product is created with primers which have two different 454 specific sequences, one called 'A' and one called 'B'. When the PCR products are then attached to the beads for emulsion PCR you have the choice of attaching either by the A primer or the B primer. Which you choose will determine which end of your PCR product will be sequenced. You CAN NOT sequence both ends of the same DNA molecule using the 454 Amplicon method. It appears that your sequencing facility prepared two different emulsion PCR reactions, one using kit A and one using kit B and then sequenced each of those. The reads in these two files are not pairs.

[plumb]

Dear kmcarr, Thank you for your reply

In our case, we have one forward and one reverse primers, but these were split into two sets with the
454 A and B adaptors switched so that sequencing would be done in both directions.

So, we think one dataset we got starts from the forward primer and one starts from the
reverse primer. Any suggestion based on those information. Thank you!

[plumb]

forget my last post.

n our case, we have one forward and one reverse primers, but these were split into two sets with the
454 A and B adaptors switched so that sequencing would be done in both directions.

Based on the above information, how should I merge the data together?

[kmcarr]

Plumb,

First of all, what is purpose of your experiment? What is the source of your input 16S RNA? Was it a population of a single species for variation analysis or was it an environmental sample for a metagenomics experiment? The purpose of your experiment will define the processing pipeline.

[plumb]

Plumb,

First of all, what is purpose of your experiment? What is the source of your input 16S RNA? Was it a population of a single species for variation analysis or was it an environmental sample for a metagenomics experiment? The purpose of your experiment will define the processing pipeline.

this is the environmental sample for metagenomics experiment. we want to measure species diversity and detailed community profile.

[kmcarr]

Plumb,

For this type of experiment (metagenomics by 16S sequencing) there is no point to sequencing the library from both the A and B primers. Since the method does not sequence the same molecule from both ends you can not combine two reads together to come up with one longer, more informative read. Look over the 16S pyrosequencing pipeline information at the Ribosomal Database Project (http://pyro.cme.msu.edu/pyro/help.jsp). They sequence their 16S V4 amplicons from just one end and then use that to assign the read based on alignment to a database of 16S genes. I'm not sure what region of the 16S you amplified but the principle is the same.

In your case I would treat the A and B read sets as if they were two different amplicons generated from the same environmental sample and see if the results from the two are consistent in terms of the community profile observed.

[HMorrison]

Dear Plumb and kmcarr,

A heads up--anyone who is doing pyrotag sequencing for community composition studies of this sort should let Roche know that they only want to sequence from one or the other end--the company is going to try to package Titanium reagents for bidirectional sequencing only, WHICH IS TOTALLY POINTLESS for this protocol since the reads can't be matched to a starting template molecules (unlike Solexa PE reads). I'm at the MBL, where the original work was done (Sogin et al., 2006), so I am familiar with this protocol and variations. As kmcarr mentions in the last reply, the only value of having an A and a B set would be as consistency check.

[454Sequencing]

Dear HMorrison,

I think I can help clarify things. It is true that the upcoming GS FLX Titanium series Amplicon emPCR kits will contain all reagents needed for generating enriched beads for bidirectional sequencing. However, the components supplied include separate 'A' and 'B' side reagents (i.e. independent A and B capture bead tubes and associated primers). This means that the kit will support a variety of sequencing strategies including the unidirectional approach of interest to you. There are sufficient reagents in a single kit to achieve this employing various experimental designs/run formats.

If you do not have a copy already, there is a Technical Bulletin on the topic of amplicon sequencing available on the my454 customer site. If you require further assistance please feel free to send a private message or contact your local Roche representative.

Best regards,
Jason

Technical Product Manager
454 Life Sciences, a Roche company

[HMorrison]

Bidirectional sequencing

That isn't what I've been hearing from Tim Harkins. Even if true, we are buying and wasting components we don't need OR we have to completely revise our experimental design.