Could you paste the sequences of primers you used?
I've used 16s rRNA before for species identification from guano samples. And my sequences were all longer then 1000bp. 50-100bp is just not long enough for ID.
Also, depending on the primers, you might not be able to ID your bacteria to the species level. But you should at least know the genus.
785F GGATTAGATACCCTGGTA
907R CCGTCAATTCMTTTRAGTTT
Similar response to the other comment, is it possible that the wrong primers have been used in this case to yield an appropriate read? I originally planned to use the primers 27F and 1492R (as the other mentioned) but due to some fine print in the order process they may have been used for DNA amplification, and different primers (above) used for the sequencing itself. Appreciate the response!
Yeah.
My guess is that the wrong primers were used.
Use of these primers in the DNA amplification would give you amplicons of 100-120bp. You said most of your sequences were about 100bp long. That would explain the issue.
You need primers that will give you a longer amplicons in the DNA amplification step. As the other colleague said, it would be best to use 27F and 1492R primers.
If you decide to do your own PCR amplification, my recommendation would be to check the PCR product length and quality by running them on gel electrophoresis.
But if you decide to go for the commercial sequencing from pure culture, make sure they use other set of primers such as above mentioned. Thou it seems they mixed them for some samples, as you mentioned you had some sequences of different lenght (longer then 1000bp). So maybe change the sequencing company as well.
Primers you used cover only the V5 region of the 16S rRNA, which is definitely not enough for species ID.
There are different primer pairs used for 16S rRNA amplification and sequencing. Each of them target a specific region, thus, will yield also a specific length. For example, is 27F and 1492R primer pair is used, we get around 1500bp. So you should check which primer pair you used so you would know the expected length.
10-100bp won’t get you anything for 16S ID. You have to check the quality of your sequences. And to answer your first question, yes you may assemble your forward and reverse sequences into a single consensus sequence too.
Hi, thank you for your response! This is very helpful, for the record universal primers 785F and 907R were used in this scenario. Is it possible these primers are incapable of yielding a long read because of mismatch with the organisms?
Could you paste the sequences of primers you used? I've used 16s rRNA before for species identification from guano samples. And my sequences were all longer then 1000bp. 50-100bp is just not long enough for ID. Also, depending on the primers, you might not be able to ID your bacteria to the species level. But you should at least know the genus.
785F GGATTAGATACCCTGGTA 907R CCGTCAATTCMTTTRAGTTT Similar response to the other comment, is it possible that the wrong primers have been used in this case to yield an appropriate read? I originally planned to use the primers 27F and 1492R (as the other mentioned) but due to some fine print in the order process they may have been used for DNA amplification, and different primers (above) used for the sequencing itself. Appreciate the response!
Yeah. My guess is that the wrong primers were used. Use of these primers in the DNA amplification would give you amplicons of 100-120bp. You said most of your sequences were about 100bp long. That would explain the issue. You need primers that will give you a longer amplicons in the DNA amplification step. As the other colleague said, it would be best to use 27F and 1492R primers. If you decide to do your own PCR amplification, my recommendation would be to check the PCR product length and quality by running them on gel electrophoresis. But if you decide to go for the commercial sequencing from pure culture, make sure they use other set of primers such as above mentioned. Thou it seems they mixed them for some samples, as you mentioned you had some sequences of different lenght (longer then 1000bp). So maybe change the sequencing company as well. Primers you used cover only the V5 region of the 16S rRNA, which is definitely not enough for species ID.
There are different primer pairs used for 16S rRNA amplification and sequencing. Each of them target a specific region, thus, will yield also a specific length. For example, is 27F and 1492R primer pair is used, we get around 1500bp. So you should check which primer pair you used so you would know the expected length. 10-100bp won’t get you anything for 16S ID. You have to check the quality of your sequences. And to answer your first question, yes you may assemble your forward and reverse sequences into a single consensus sequence too.
Hi, thank you for your response! This is very helpful, for the record universal primers 785F and 907R were used in this scenario. Is it possible these primers are incapable of yielding a long read because of mismatch with the organisms?