Read lenght in 16S RNA data

k_a_r_o_l · 04-11-2017, 07:39 AM

Hi,

My amplicon after assembly shoud have approx. 460bp. Do I have to analyze only 460bp products or can I also analyze some shorter/longer amplicons. If yes, how to set a specific lenght range/limit. For example I have 200000 reads per sample, almost 52% of them are shorter than 460bp, but only 11% are shorter than 440bp. I think that those reads also have an importance and value, but how to set the range?

Please help me.

fanli · 04-11-2017, 09:27 AM

What amplicon are you using? Some of them have substantial variation in length in the variable region.

See:
https://www.biostars.org/p/78179/

k_a_r_o_l · 04-11-2017, 09:37 AM

primes that target 16S V3 and V4 region (used in illumina 16S metagenomic protocol)

thermophile · 04-11-2017, 09:54 AM

I pretty strongly recommend using mothur which aligned the reads to a know database (more robust to discard seqs that don't align rather than picking an arbitrary length). But, know that the illumina 16s kit produces not great results because you have so little overlap to correct miscalled bases.

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04-11-2017, 07:39 AM	#1
k_a_r_o_l Member Location: Warsaw Join Date: Mar 2014 Posts: 17	Read lenght in 16S RNA data Hi, My amplicon after assembly shoud have approx. 460bp. Do I have to analyze only 460bp products or can I also analyze some shorter/longer amplicons. If yes, how to set a specific lenght range/limit. For example I have 200000 reads per sample, almost 52% of them are shorter than 460bp, but only 11% are shorter than 440bp. I think that those reads also have an importance and value, but how to set the range? Please help me.

04-11-2017, 09:54 AM	#4
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	I pretty strongly recommend using mothur which aligned the reads to a know database (more robust to discard seqs that don't align rather than picking an arbitrary length). But, know that the illumina 16s kit produces not great results because you have so little overlap to correct miscalled bases. __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

04-11-2017, 09:27 AM	#2
fanli Senior Member Location: California Join Date: Jul 2014 Posts: 198	What amplicon are you using? Some of them have substantial variation in length in the variable region. See: https://www.biostars.org/p/78179/

04-11-2017, 09:37 AM	#3
k_a_r_o_l Member Location: Warsaw Join Date: Mar 2014 Posts: 17	primes that target 16S V3 and V4 region (used in illumina 16S metagenomic protocol)