Hi,all.I am dealing with some 16S rRNA data from Miseq platform.They are 2*300 reads.

I use Flash software to merge them with the parameter -M 220 directly.And UPARSE is used to filter

the low quality reads with the paramater -maxee 1.

Here is the quality of one of the sample:

read1:

#Base     Mean    Median   Mean    Median
215-219   33.45   38       20.24   22.8
220-224   33.98   38       19.33   21.6
225-229   33.37   38       18.73   21.2
230-234   32.64   37.4     16.15   12.8
235-239   33.15   38       13.53   3.2
240-244   32.93   37.8     12.03   2
245-249   31.70   37       11.13   2
250-254   31.45   36.6     10.12   2
255-259   30.92   36.6     8.31    2
260-264   29.69   35.2     7.47    2
265-269   30.21   36.8     6.55    2
270-274   28.26   34.8     5.78    2
275-279   29.06   36.4     5.12    2
280-284   27.60   34.4     4.37    2
285-289   26.12   31.4     3.86    2
290-294   25.17   31       3.14    2
295-299   24.63   32.2     2.76    2
300-301   21.48   28       2.39    2

read2:

#Base     Mean    Median   Mean   Median
210-214   21.20   24.2     2      35      2   37.6
215-219   20.24   22.8     2      34.6    2   37.2
220-224   19.33   21.6     2      34      2   37
225-229   18.73   21.2     2      34.6    2   37.2
230-234   16.15   12.8     2      31.4    2   36.8
235-239   13.53   3.2      2      27      2   35
240-244   12.03   2        2      24.2    2   33
245-249   11.13   2        2      22.4    2   33
250-254   10.12   2        2      20.2    2   32.4
255-259   8.31    2        2      12      2   28.8
260-264   7.47    2        2      4.2     2   27.6
265-269   6.55    2        2      2       2   25.6
270-274   5.78    2        2      2       2   22.8
275-279   5.12    2        2      2       2   19.2
280-284   4.37    2        2      2       2   8.2
285-289   3.86    2        2      2       2   2
290-294   3.14    2        2      2       2   2
295-299   2.76    2        2      2       2   2
300-301   2.39    2        2      2       2   2

At last I get 3125518 reads.After dereplication 2557464 retained include 2409776 singletons(A singleton is a read with a sequence that is present exactly once, i.e. is unique among the reads). Is that too much? After all they have been amplificated sever times.

I follow this pipeline(http://drive5.com/usearch/manual/uparse_cmds.html) to continue the process.At last I have 1321653 in 3142 OTUs.

Anything wrong while I processing the data or this percent just normal.

Thanks for response