Sam’s Notebook: Data Received – Additional C.gigas Whole Genome Bisulfite Sequencing Data from Genewiz

The FastQC analysis of the intitial data we received from Genewiz (on 20190408)showed consistent failures in the “Per Tile Sequence Quality” for all of Roberto’s Crassostrea gigas sequencing. After discussing with Genewiz, they offered to generate an additional 25% reads for each of those libraries.

The data became available today. The additional reads were appended to the previous sequencing results, so the filenames remain the same as before.

Data was downloaded to owl/nightingales/C_gigas:

http://bit.ly/1C2LBad

Roberto’s samples match the following filename pattern:

[035]*.gz

Will generate new FastQC analysis for these files. Since the additional data was simply appended to the previous data, I fully expect the “Per Tile Sequence Quality” to fail again. However, this additional data should help compensate for data loss we will experience after quality trimming.

Updated sequencing report:

Table 2.1 Sample Sequencing Statistics
GENEWIZ NGS Data Report
Project Sample ID Barcode Sequence # Reads Yield (Mbases) Mean Quality Score % Bases >= 30
30_183897003 Tank2-025-026 GCCAAT 66223383 19867 37.04 87.98
30_183897003 0502 CAGATC 62707839 18813 35.43 82.96
30_183897003 5201 ACAGTG 60125207 18037 35.67 83.78
30_183897003 3503 CCGTCC 59836967 17951 35 81.35
30_183897003 5902 AGTTCC 64657824 19397 35.43 83.09
30_183897003 5202 AGTCAA 54986647 16496 35.45 83.07
30_183897003 3501 TGACCA 59575535 17873 35.57 83.51
30_183897003 YRVL TGACCA 66590575 19977 37.05 87.99
30_183897003 3502 CTTGTA 62881856 18864 35.51 83.18
30_183897003 5903 GTGAAA 54173403 16252 37.06 88.07
30_183897003 0503 ATGTCA 60779896 18234 35.24 82.18
30_183897003 YRVA CGATGT 67542867 20263 37.1 88.14
30_183897003 Tank3-15-16 ACAGTG 49500297 14850 36.96 87.73
30_183897003 0501 CGATGT 69631899 20890 35.42 83.2
30_183897003 5203 GTCCGC 63065336 18920 35.12 81.9
30_183897003 5901 GCCAAT 72859227 21858 35.42 82.92

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Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemolymph Pellet in RNAlater

After the success we had isolating RNA using the Quick-DNA/RNA Microprep Plus Kit (ZymoResearch), Steven had me isolate RNA from a list of ~117 samples. Of that list, I was able to find 66 crab hemolymph pelleted RNAlater samples. The “missing” samples were most likely previosly used by Grace during our various attempts to get some usable RNA out these.

Used 70uL of each RNAlater/hemolymph “slurry”. This was intended to maximize the sample size and speed of the procedure. If I had done calculations correctly, it would have meant not having multiple round of transferring the samples to the columns. However, I should have used 35uL! So, this is just a note to my future self…

Followed the Zymo protocol for “Samples in RNAlater” (used H2O as intial sample diluent) and included the on-column DNase step.

Eluted with 15uL.

Quantified RNA using the Roberts Lab Qubit 3.0 and the RNA High Sensitivity Assay (Invitrogen).

Used 2uL of each sample.

NOTE: Sample #82 did not look like all the others. It was clear, while all other samples were cloudy and kind of thicker in consistency.:

Comparison of clear and hazy crab hemo pellet in RNAlater