Trimming and Quality filtering of 2bRAD data

Here at my github is a markdown file with the code used to trim and quality filter the 2bRAD sequencing data from December 2015.


Size & 2bRAD Read #’s for MBD BSseq Samples

18 of the adult Olympia oyster broodstock samples that were 2bRAD sequenced were also sent off for MBD-enriched and bisulfite treatment sequencing to get epigenetic information (see project wiki). We wanted to associate size data with these samples and check if the 2brad sequencing worked.

First, some correction of sample names in the 2brad demultiplexing:

When I was on the last step of the 2brad protocol I accidentally deleted some of the sample names in Library 3 from the “Library” sheet in the master sample sheet. I didn’t notice until making the barcode sample sheet and so denoted those on the barcode sheet by putting “w” next to the name. I went back in the sheet revision history to 11/21/15 at 3:22pm and associated the correct name with the well and barcode for these and checked all other samples.

  • HC4_12w -> HC4_11
  • HC4_13w -> HC4_10
  • HC4_15w -> HC4_15
  • HC4_1w -> HC4_7
  • HC4_5w -> HC4_8
  • HC5_1w -> HC5_1
  • SS3_15w -> SS3_20
  • SS3_16w -> SS3_21
  • SS4_1Aw -> SS4_7
  • SS4_1Bw -> SS4_1
  • SS4_3w -> SS4_16
  • SS4_7w -> SS4_9
  • SS4_9 -> SS4_3


When the broodstock oysters were dissected at Manchester last summer, I weighed them in the shell and took pictures of them first with a ruler so later they could be measured (dissections done on 8/13/15 and 8/17/15). I took the pictures for families HC1,  HC2, HC3, SS2, SS3, and SS5 and opened them in ImageJ. For each picture, I set the scale by drawing a line on 1 cm of the ruler to get the number of pixels per mm 3 times and getting the average pixels/mm. To get shell width, I drew a line between the two widest dorsal-ventral points of the shell (with the hinge always on bottom. I did this 2x for each oyster and took the average of the measurements.

Screenshot from 2016-02-12 16:28:19

I also measured shell area by drawing a line around the oyster shell 2x and taking the average. These obviously have more spread in the values.

Screenshot from 2016-02-12 16:33:26

Read Numbers






Sample Weight (grams in shell) Length (mm) Raw 2bRAD reads
hc1_2B 2.2 17.41 1521
hc1_4B 1.9 20.43 2294290
hc2_15B 2.2 25.33 2498
hc2_17 1.1 19.38 (seq 4x)best = 2964986
hc3_1 2.2 26.79 1863108
hc3_5 1.9 19.5 1915
hc3_7 1.4 18.43 2438612
hc3_10 1.2 19.80 4091281
hc3_11 2.1 20.54 2901608
ss2_9B 1.5 17.2 749
ss2_14B 2.2 21.02 29745
ss2_18B 5.3 35.76 3016
ss3_3B 2.1 22.71 2076769
ss3_14B 2.4 24.71 2661152
ss3_15B 2.6 26.78 2816364
ss3_16B 4.2 39.57 2070520
ss3_20 2.3 26.96 1656456
ss5_18 5.4 27.34 2358172