Laura’s Notebook: Oly RNA – Data processing test-run

Things to note about the QuantSeq libraries:

  • I used the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
  • Reads are therefore stranded – generated from forward (FWD) strand
  • The kit does not enrich for poly(A) or ribosomal RNA (rRNA) before 1st strand synthesis
  • 1st strand synthesis occurs near the 3’ (poly(a)) end of the strand via oligodT priming. Therefore reads are more likely to contain the 3’ end of the sequence. Also, reads are less likely to contain exon junction sites, and are not randomly distributed across the mRNA sequence. Thus, data does not likely contain isoform information (does not represent the whole gene), and is more likely to appear as “duplicated” since reads are more likely to be pulled from the same start location.
  • Library insert lengths averaged ~250bp, but sequencing length was 100bp. Since adapter sequences & poly-a tails can be up to ~25 total, all raw reads contain ~75-85bp of actual mRNA sequence information.
  • Sequencing was performed on a NovaSeq, and was single-end.

Data processing test-run on 8 samples

I’m developing my pipeline based on 8 of the total 144 samples. Of the 8 test samples, 2 are from adult gill, 2 from juvenile whole-body, and 4 are from larval samples (sample numbers are 141, 159, 302m, 331, 43, 441, 483, and 563). Here is my bioinformatics pipeline.

NOTE: for each of STAR, Bowtie2, and Salmon I tested various combinations of settings to try to optimize alignment rate. More details on that later.

Bioinformatics-process1Bioinformatics-process2Bioinformatics-process3Bioinformatics-process4Bioinformatics-process5

Some notes on mapping QC

  • % mapped reads – higher when mapping to genome. E.g. for human samples, 70-90% of reads typically map to human genome (lower mapping rate expected for transcriptomes).
  • k-mer and GC content may reveal PCR biases. k-mer and GC content varies by species/experiment, BUT should be homogenous across samples in the same experiment
  • Could check if rRNA and small RNAs are present – should not be. R packages NOISeq or EDASeq provide useful plots for QC of count data.
  • Could try Picard for mapping quality control. Try using the CollectMultipleMetrics function. This does the following:

Collect multiple classes of metrics. This ‘meta-metrics’ tool runs one or more of the metrics collection modules at the same time to cut down on the time spent reading in data from input files. Available modules include CollectAlignmentSummaryMetrics, CollectInsertSizeMetrics, QualityScoreDistribution, MeanQualityByCycle, CollectBaseDistributionByCycle, CollectGcBiasMetrics, RnaSeqMetrics, CollectSequencingArtifactMetrics, and CollectQualityYieldMetrics.

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Laura’s Notebook: Oly OA RNA isolation – raw data

Erica at the NWGC worked very quickly to get my samples sequenced on the NovaSeq. On Monday she pooled them and ran each pool on the Bioanalyzer. Here is the bioanalyzer report, and a note from Erica about the pool concentrations:

Here is the QC: I did dilute an aliquot of the pool down further to run the samples better. The original qubit pools were as follows and these were what was loaded on the bioanalyzer.

  • Batch1_69plex 10.966ng/ul
  • Batch2_77plex 11.649ng/ul

The diluted qubit values are:

  • Batch1_69plex 2.777ng/ul
  • Batch2_77plex 2.43ng/ul

Once the samples were finished on the NovaSeq, she sent me a summary of the run, and files for each pool with the # reads per sample:

Date Lane Lane Concentration (pM) SAV Clusters/mm^2 % Clusters PF Overall Q30 Read 1 Q30
200420 1 Batch1_69plex 270 506.12 79.29 89.73 88.93
2 Batch2_77plex

I received a link to the raw data, which is not yet demultiplexed (I will need to do that). The data is stored on Globus.org. I will need to transfer it from there to the Nightengales directory on Owl in the O. lurida folder, which is where all raw NGS data is housed for the Roberts Lab. This could be done in a few ways, but I will follow Shelly’s lead, with a few changes based on my work-at-home situation. She directed me to this notebook post, and this github issue as references. Globus endpoints are set up via a GUI, so since I am working at home I opted to tranfer the files to my external hard drive, and then to other locations.

Set up Globus personal endpoint, download data

Following these Globus setup instructions I set my laptop up as a personal endpoint on Globus. I edited my Globus settings to be able to write to my external hard drive. I then transferred the two FASTQ files with their md5 files to my external hard drive.

image

image

Mount Owl, transfer files to Nightengales

I mounted Owl on my computer using Finder –> Go –> Connect to Server. I entered owl’s address (afp://owl.fish.washington.edu), then my username and pw.

To be continued…

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Laura’s Notebook: Oly OA RNA isolation – sequencing

I’m done with my QuantSeq libraries! After getting a few quotes for sequencing, we’ve decided have the UW Genome Sciences sequencing core do it (the Sequencing Northwest Genomics Center). They also pooled my libraries for me for a small fee, but I needed to provide them with library concentrations (from QuBit). I also sent them my BioAnalyzer results (mean library length).

On Friday, April 17 I prepped my samples and walked them over to Erica at the genome science center. Here’s what needed to be done:

Quantified a few more libraries using Qubit.

I had to quantify my juvenile O. lurida libraries, and a couple random libraries that I previously omitted. I used the Qubit 1X dsDNA HS Assay.

Transerred libraries to new PCR plates

I submitted 8 uL of each library, which is ~half of my volumes (started with ~16 uL). I also wanted to make sure the sequencing facility would correctly pool my libraries in two batches – 1) Ctenidia+Juvenile and 2) Larvae. This is because some of my index numbers were the same in Batch 1 as in Batch 2, so they needed to be run on separate lanes. To make this very clear, I prepared 2 PCR plates, one for each batch/lane. Also, because I re-prepped a few libraries, I needed to carefully identify which to transfer, and which to omit.

image

Library details

Below are details for the libraries I submitted, with information such as original RNA concentration used for library prep, # PCR cycles used to amplify libraries, library concentration (DNA, from Qubit), mean library length (for a few), index number. All this information is located in this Sample-Submission-and-Info.xlsx spreadsheet.

Here is the manifest that I sent to Erica: Spencer,LH_Sample-Manifest-2020-04-17.xlsx

Here is the sample submission sheet: Spencer,LH_Sample-Submission-Form-2020-04-17.pdf

A note on PhiX Spike-in:

Erica asked whether I would like PhiX spike-in. Standard protocol for any Illumina platform is to do a 1% spike-in for quality control. The QuantSeq library prep manual does not specify whether it is recommended for my library type, although it does specify for QuantSeq libraries prepped using an add-on module which tack on UMIs to each library. I eailed my QuantSeq rep, and they replied quickly with the following: “For standard QuantSeq libraries without UMIs, 1% is fine. The reason libraries with UMIs need more is due to the spacer we use with the UMIs. It has a unique fingerprint that requires a more PhiX. That is the only reason. Even when labs combine non-UMI libraries with UMI libraries, they no longer need the higher percentage of PhiX. It is for UMI only libraries in a flow cell lane. It is rather specific.” So, in the end I had them use a 1% PhiX spike-in.

Adult O. lurida ctenidia samples, multiple populations and pCO2 exposures

Plate/Batch/Lane # Well Sample No. [RNA] (ng/ul) Cycles, round down (for most) Tissue source Tissue type [DNA] (ng/uL) Bioanalyzer mean bp INDEX # Index bp sequence Population Parental pCO2
1 D06 291 158.0 15 Adult ctenidia 3.54 NT 7035 GTTACC Dabob Bay High
1 C08 292 29.6 15 Adult ctenidia 1.32 NT 7037 TGGCGA Dabob Bay High
1 A11 293 39.6 16 Adult ctenidia 1.03 NT 7014 AATCCG Dabob Bay High
1 A07 294 110.0 16 Adult ctenidia 1.95 245 7008 TGTGCA Dabob Bay High
1 D05 295 34.8 15 Adult ctenidia 3.58 NT 7050 TCGAGG Dabob Bay High
1 D04 296 180.0 15 Adult ctenidia 3.46 NT 7046 CTCCAT Dabob Bay High
1 C01 298 182.0 15 Adult ctenidia 3.08 NT 7028 GCTCGA Dabob Bay High
1 B09 299 50.4 15 Adult ctenidia 2.46 NT 7024 CCGCAA Dabob Bay High
1 B10 301 75.8 15 Adult ctenidia 2.82 NT 7025 TTTATG Dabob Bay Ambient
1 B03 302 62.4 14 Adult ctenidia 4.9 NT 7018 GTCAGG Dabob Bay Ambient
1 B05 303 95.2 14 Adult ctenidia 2.4 NT 7020 TATGTC Dabob Bay Ambient
1 B12 304 200.0 15 Adult ctenidia 2.8 NT 7027 CAAGCA Dabob Bay Ambient
1 A01 305 75.2 16 Adult ctenidia 2.96 267 7002 GATCAC Dabob Bay Ambient
1 D08 306 136.0 15 Adult ctenidia 4.14 NT 7051 CACTAA Dabob Bay Ambient
1 D09 307 89.4 15 Adult ctenidia 3.32 NT 7053 CGCCTG Dabob Bay Ambient
1 A05 308 73.6 16 Adult ctenidia 2.48 256 7006 GTGTAG Dabob Bay Ambient
1 B08 309 170.0 14 Adult ctenidia 1.46 NT 7023 CACACT Dabob Bay Ambient
1 A02 311 158.0 16 Adult ctenidia 3.76 262 7003 ACCAGT Oyster Bay C1 High
1 C04 312 90.6 15 Adult ctenidia 1.58 NT 7032 CGAAGG Oyster Bay C1 High
1 C12 313 72.4 15 Adult ctenidia 1.81 NT 7041 CTCTCG Oyster Bay C1 High
1 E01 314 42.2 17 Adult ctenidia 4.32 NT 7049 GTGCCA Oyster Bay C1 High
1 C02 315 148.0 15 Adult ctenidia 2.32 NT 7029 GCGAAT Oyster Bay C1 High
1 A09 316 146.0 16 Adult ctenidia 3.74 252 7012 ATGAAC Oyster Bay C1 High
1 C09 317 158.0 15 Adult ctenidia 2.14 NT 7038 ACCGTG Oyster Bay C1 High
1 A06 318 174.0 16 Adult ctenidia 4.08 262 7007 CTAGTC Oyster Bay C1 High
1 E04 319 77.6 17 Adult ctenidia 3.02 NT 7011 TTAACT Oyster Bay C1 High
1 C05 321 148.0 15 Adult ctenidia 3.64 NT 7033 AGATAG Oyster Bay C1 Ambient
1 A08 322 44.6 16 Adult ctenidia 2.7 269 7010 CGGTTA Oyster Bay C1 Ambient
1 D03 323 102.0 15 Adult ctenidia 3.7 NT 7044 ACAGAT Oyster Bay C1 Ambient
1 D11 324 172.0 16 Adult ctenidia 2.84 NT 7009 TCAGGA Oyster Bay C1 Ambient
1 B01 325 180.0 14 Adult ctenidia 2.92 NT 7016 TACCTT Oyster Bay C1 Ambient
1 D02 326 130.0 15 Adult ctenidia 4.04 NT 7043 AAGACA Oyster Bay C1 Ambient
1 D01 327 85.2 15 Adult ctenidia 1.97 NT 7042 TGACAC Oyster Bay C1 Ambient
1 A12 328 156.0 14 Adult ctenidia 2.12 NT 7015 GGCTGC Oyster Bay C1 Ambient
1 D10 329 162.0 16 Adult ctenidia 5.1 NT 7045 TAGGCT Oyster Bay C1 Ambient
1 E05 331 42.2 19 Adult ctenidia 9.6 NT 7001 CAGCGT Fidalgo Bay High
1 D12 332 65.8 16 Adult ctenidia 2.14 NT 7047 GCATGG Fidalgo Bay High
1 C06 333 78.6 15 Adult ctenidia 2.98 NT 7034 TTGGTA Fidalgo Bay High
1 E02 334 64.8 17 Adult ctenidia 3.92 NT 7048 AATAGC Fidalgo Bay High
1 C07 335 180.0 15 Adult ctenidia 2.58 NT 7036 CGCAAC Fidalgo Bay High
1 E03 336 94.8 17 Adult ctenidia 4.6 NT 7052 GGTATA Fidalgo Bay High
1 C11 337 194.0 15 Adult ctenidia 4.02 NT 7040 GATTGT Fidalgo Bay High
1 A04 338 81.6 16 Adult ctenidia 2.74 269 7005 ACATTA Fidalgo Bay High
1 A10 339 77.2 16 Adult ctenidia 1.91 NT 7013 CCTAAG Fidalgo Bay High
1 B07 341 89.6 14 Adult ctenidia 1.31 NT 7022 GGAGGT Fidalgo Bay Ambient
1 B11 342 162.0 15 Adult ctenidia 1.78 NT 7026 AACGCC Fidalgo Bay Ambient
1 B02 343 114.0 14 Adult ctenidia 2.58 NT 7017 TCTTAA Fidalgo Bay Ambient
1 C03 344 25.0 15 Adult ctenidia 1.58 NT 7030 TGGATT Fidalgo Bay Ambient
1 B04 345 190.0 14 Adult ctenidia 3.5 NT 7019 ATACTG Fidalgo Bay Ambient
1 B06 346 43.6 14 Adult ctenidia 1.36 NT 7021 GAGTCC Fidalgo Bay Ambient
1 D07 347 69.0 15 Adult ctenidia 4.36 NT 7031 ACCTAC Fidalgo Bay Ambient
1 A03 348 54.4 16 Adult ctenidia 3.06 265 7004 TGCACG Fidalgo Bay Ambient
1 C10 349 82.0 15 Adult ctenidia 1.9 NT 7039 CAACAG Fidalgo Bay Ambient

Juvenile O. lurida whole-body samples from those deployed in Port Gamble, with two populations and two parental pCO2 exposures

Plate/Batch/Lane # Well Sample No. [RNA] (ng/ul) Cycles, round down (for most) Tissue source Tissue type [DNA] (ng/uL) Bioanalyzer mean bp INDEX # Index bp sequence Population Parental pCO2
1 G02 137 99.2 15 Juvenile whole body, individual 5.2 NT 7090 CCTGCT Dabob Bay Ambient
1 F01 139 136.0 14 Juvenile whole body, individual 13.3 294 7081 GCAGCC Dabob Bay Ambient
1 G03 140 174.0 15 Juvenile whole body, individual 7.62 314 7091 GCGCTG Dabob Bay Ambient
1 F08 141 190.0 15 Juvenile whole body, individual 23.6 NT 7088 AGACCA Dabob Bay Ambient
1 G05 156 110.0 16 Juvenile whole body, individual 4.32 NT 7093 TTCGAG Fidalgo Bay Ambient
1 G07 159 184.0 16 Juvenile whole body, individual 23 283 7095 AGGCAT Fidalgo Bay Ambient
1 F04 161 168.0 14 Juvenile whole body, individual 6.22 290 7084 AAGTGG Fidalgo Bay Ambient
1 G01 162 164 15 Juvenile whole body, individual 6.74 289 7080 AACAAG Fidalgo Bay Ambient
1 G06 168 89.0 16 Juvenile whole body, individual 15.9 305 7094 AGAATC Dabob Bay High
1 G04 169 95.6 16 Juvenile whole body, individual 3.68 310 7092 GAACCT Dabob Bay High
1 F05 171 146.0 15 Juvenile whole body, individual 3.22 305 7085 CTCATA Dabob Bay High
1 F06 172 116.0 15 Juvenile whole body, individual 10.6 NT 7086 CCGACC Dabob Bay High
1 G08 181 138.0 16 Juvenile whole body, individual 2.84 280 7096 ACACGC Fidalgo Bay High
1 F03 183 196.0 14 Juvenile whole body, individual 5.88 NT 7083 TGCTAT Fidalgo Bay High
1 F02 184 96.4 14 Juvenile whole body, individual 6.1 NT 7082 ACTCTT Fidalgo Bay High
1 F07 185 102.0 15 Juvenile whole body, individual 15 296 7087 GGCCAA Fidalgo Bay High

Larval O. lurida, multiple cohorts, parental pCO2 and temperature exposures. Larvae are pooled by pulse/family.

Plate/Batch/Lane # Well Sample No. [RNA] (ng/ul) Vol RNA used Cycles, round down (for most) Tissue source Tissue type [DNA] (ng/uL) Bioanalyzer mean bp INDEX # Index bp sequence Population Parental Temperature Parental pCO2 Larval sample #
2 F08 34 104.0 3.37 19 Larval whole body, pooled 3.8 NT 7073 GACATC Oyster Bay C1 6 Ambient 77-A
2 F12 35 94.4 3.71 21 Larval whole body, pooled 0.522 NT 7075 CGTCGC Oyster Bay C1 6 Ambient 10-A
2 F10 37 74.6 4.69 21 Larval whole body, pooled 5.46 NT 7076 ATGGCG Oyster Bay C1 6 Ambient 69-A
2 E04 39 89.8 3.90 15 Larval whole body, pooled 2.04 NT 7056 ATATCC Oyster Bay C1 6 Ambient 48-A
2 F02 41 108.0 3.24 19 Larval whole body, pooled 6.82 NT 7068 CCAATT Oyster Bay C1 10 High 06-A
2 D10 43 156.0 2.24 15 Larval whole body, pooled 2.48 261 7050 TCGAGG Oyster Bay C1 10 High 08-A
2 F11 44 41.4 5.00 21 Larval whole body, pooled 0.18 NT 7078 GCCACA Oyster Bay C1 10 High 32-A
2 F03 45 25.1 5 19 Larval whole body, pooled 4.8 NT 7077 ATTGGT Oyster Bay C1 10 High 79-A
2 F06 46 98.6 3.55 19 Larval whole body, pooled 4.52 NT 7074 CGATCT Oyster Bay C1 10 High 24-A
2 E10 47 128.0 2.73 16 Larval whole body, pooled 2.58 NT 7070 AACCGA Oyster Bay C1 10 High 26-A
2 A10 401 93.4 3.75 16 Larval whole body, pooled 1.24 325 7012 ATGAAC Dabob Bay 10 Ambient 14-A
2 A05 402 114.0 3.07 16 Larval whole body, pooled 2.76 346 7006 GTGTAG Dabob Bay 10 Ambient 31-A
2 C06 403 136.0 2.57 14 Larval whole body, pooled 4.16 NT 7032 CGAAGG Dabob Bay 10 Ambient 75-A
2 D06 404 112.0 3.13 15 Larval whole body, pooled 2.92 NT 7046 CTCCAT Dabob Bay 10 Ambient 80-A
2 A09 411 72.6 4.82 16 Larval whole body, pooled 3.08 288 7011 TTAACT Dabob Bay 10 High 23-A
2 G02 412 31.2 5.00 23 Larval whole body, pooled 2.52 NT 7069 AGTTGA Dabob Bay 10 High 27-A
2 D02 413 130.0 2.69 15 Larval whole body, pooled 1.97 NT 7041 CTCTCG Dabob Bay 10 High 58-A
2 E01 414 168.0 2.08 15 Larval whole body, pooled 0.858 NT 7053 CGCCTG Dabob Bay 10 High 60-A
2 B10 421 57.6 5.00 14 Larval whole body, pooled 1.63 NT 7024 CCGCAA Dabob Bay 6 Ambient 59-A
2 G03 432 74.0 4.73 23 Larval whole body, pooled 6.28 NT 7065 AAGCTC Dabob Bay 6 High 72-A
2 E07 434 162.0 2.16 15 Larval whole body, pooled 5.58 NT 7059 GGTGAG Dabob Bay 6 High 74-A
2 G01 441 16.2 5.00 23 Larval whole body, pooled 0.866 NT 7061 GAAGTG Fidalgo Bay 10 Ambient 20-A
2 C12 443 60.2 5.00 15 Larval whole body, pooled 1.36 NT 7039 CAACAG Fidalgo Bay 10 Ambient 53-A
2 C10 444 70.6 4.96 14 Larval whole body, pooled 4.08 NT 7037 TGGCGA Fidalgo Bay 10 Ambient 63-A
2 D09 445 160.0 2.19 15 Larval whole body, pooled 4 NT 7049 GTGCCA Fidalgo Bay 10 Ambient 65-A
2 C04 451 68.4 5.12 14 Larval whole body, pooled 2.24 NT 7030 TGGATT Fidalgo Bay 10 High 16-A
2 A12 453 196.0 1.79 14 Larval whole body, pooled 0.968 NT 7014 AATCCG Fidalgo Bay 10 High 36-A
2 B08 473 124.0 2.82 14 Larval whole body, pooled 2.34 NT 7022 GGAGGT Fidalgo Bay 6 High 46-A
2 B02 474 77.2 4.53 14 Larval whole body, pooled 3.02 NT 7016 TACCTT Fidalgo Bay 6 High 47-A
2 B04 475 27.4 5.00 14 Larval whole body, pooled 2.44 NT 7018 GTCAGG Fidalgo Bay 6 High 50-A
2 B05 476 39.2 5.00 14 Larval whole body, pooled 4.24 NT 7019 ATACTG Fidalgo Bay 6 High 54-A
2 D01 477 164.0 2.13 15 Larval whole body, pooled 2.28 NT 7040 GATTGT Fidalgo Bay 6 High 76-A
2 E09 481 89.2 3.92 15 Larval whole body, pooled 2.18 NT 7036 CGCAAC Oyster Bay C1 10 Ambient 02-A
2 C01 482 22.2 5.00 14 Larval whole body, pooled 1.42 NT 7027 CAAGCA Oyster Bay C1 10 Ambient 04-A
2 A07 483 99.0 3.54 16 Larval whole body, pooled 1.22 219 7009 TCAGGA Oyster Bay C1 10 Ambient 03-A
2 D07 484 58.4 5.00 15 Larval whole body, pooled 1.67 NT 7047 GCATGG Oyster Bay C1 10 Ambient 09-A
2 B09 485 19.1 5.00 14 Larval whole body, pooled 2.9 NT 7023 CACACT Oyster Bay C1 10 Ambient 34-A
2 A06 487 118.0 2.97 16 Larval whole body, pooled 1.27 290 7007 CTAGTC Oyster Bay C1 10 Ambient 40-A
2 C02 488 60.0 5.00 14 Larval whole body, pooled 3.06 NT 7028 GCTCGA Oyster Bay C1 10 Ambient 44-A
2 C11 489 68.0 5.00 15 Larval whole body, pooled 3.84 272 7038 ACCGTG Oyster Bay C1 10 Ambient 49-A
2 B07 490 186.0 1.88 14 Larval whole body, pooled 2.32 NT 7021 GAGTCC Oyster Bay C1 10 Ambient 64-A
2 E02 491 58.4 5.00 15 Larval whole body, pooled 6.64 NT 7054 AATGAA Oyster Bay C1 10 Ambient 66-A
2 D04 492 82.6 4.24 15 Larval whole body, pooled 2.02 NT 7043 AAGACA Oyster Bay C1 10 Ambient 81-A
2 D11 506 29.2 5.00 15 Larval whole body, pooled 1.63 NT 7051 CACTAA Oyster Bay C1 10 High 62-A
2 E08 513 142.0 2.46 15 Larval whole body, pooled 5.92 NT 7060 TTCCGC Oyster Bay C1 6 Ambient 45-A
2 F05 521 66.6 5.00 19 Larval whole body, pooled 8.66 NT 7071 CAGATG Oyster Bay C1 6 High 01-A
2 A01 522 32.2 5.00 16 Larval whole body, pooled 2.42 NT 7002 GATCAC Oyster Bay C1 6 High 07-A
2 A04 523 71.4 4.90 16 Larval whole body, pooled 2.16 276 7005 ACATTA Oyster Bay C1 6 High 25-A
2 A11 524 63.0 5.00 14 Larval whole body, pooled 3.24 NT 7013 CCTAAG Oyster Bay C1 6 High 28-A
2 B06 525 140.0 2.50 14 Larval whole body, pooled 2.44 NT 7020 TATGTC Oyster Bay C1 6 High 30-A
2 B03 526 138.0 2.54 14 Larval whole body, pooled 2.2 NT 7017 TCTTAA Oyster Bay C1 6 High 33-A
2 D03 527 124.0 2.82 15 Larval whole body, pooled 1.42 243 7042 TGACAC Oyster Bay C1 6 High 61-A
2 C03 528 87.6 4.00 14 Larval whole body, pooled 4.48 NT 7029 GCGAAT Oyster Bay C1 6 High 68-A
2 E05 529 56.8 5.00 15 Larval whole body, pooled 2.86 NT 7057 AGTACT Oyster Bay C1 6 High 70-A
2 D08 531 95.4 3.67 15 Larval whole body, pooled 2.1 NT 7048 AATAGC Oyster Bay C2 10 Ambient 17-A
2 C09 532 93.6 3.74 14 Larval whole body, pooled 2.12 NT 7035 GTTACC Oyster Bay C2 10 Ambient 42-A
2 F01 533 6.5 5.00 17 Larval whole body, pooled 5.36 NT 7063 ACGTCT Oyster Bay C2 10 Ambient 56-A
2 D05 541 44.4 5.00 15 Larval whole body, pooled 1.4 NT 7044 ACAGAT Oyster Bay C2 10 High 12-A
2 A03 542 32.8 5.00 16 Larval whole body, pooled 2.22 270 7004 TGCACG Oyster Bay C2 10 High 13-A
2 C05 543 162.0 2.16 14 Larval whole body, pooled 5.24 NT 7031 ACCTAC Oyster Bay C2 10 High 43-A
2 B12 551 96.4 3.63 14 Larval whole body, pooled 1.69 NT 7026 AACGCC Oyster Bay C2 6 Ambient 35-A
2 B11 553 186.0 1.88 14 Larval whole body, pooled 3.28 NT 7025 TTTATG Oyster Bay C2 6 Ambient 55-A
2 B01 554 188.0 1.86 14 Larval whole body, pooled 2.38 NT 7015 GGCTGC Oyster Bay C2 6 Ambient 78-A
2 F09 561 28.0 5.00 21 Larval whole body, pooled 4.1 NT 7066 GACGAT Oyster Bay C2 6 High 05-A
2 G05 562 126 2.777778 23 Larval whole body, pooled 9.88 NT 7064 CAGGAC Oyster Bay C2 6 High 11-A
2 E11 563 47.0 5.00 16 Larval whole body, pooled 4.66 NT 7045 TAGGCT Oyster Bay C2 6 High 15-A
2 C08 564 152.0 2.30 14 Larval whole body, pooled 2.24 NT 7034 TTGGTA Oyster Bay C2 6 High 52-A
2 F07 565 31.2 5.00 19 Larval whole body, pooled 3.2 NT 7067 TCGTTC Oyster Bay C2 6 High 57-A
2 G04 571 LOW 5.00 23 Larval whole body, pooled 0.232 287 7072 GTAGAA Negative control NA NA NA
2 A08 431b 83.0 4.22 16 Larval whole body, pooled 3.36 285 7010 CGGTTA Dabob Bay 6 High 51-A
2 E12 442b 69.8 5.00 17 Larval whole body, pooled 3.2 NT 7062 CAATGC Fidalgo Bay 10 Ambient 38-A
2 A02 452b 97.2 3.60 16 Larval whole body, pooled 2.68 294 7003 ACCAGT Fidalgo Bay 10 High 18-A
2 C07 461b 84.0 4.17 14 Larval whole body, pooled 2.02 NT 7033 AGATAG Fidalgo Bay 6 Ambient 22-A
2 F04 462b 106 3.301887 19 Larval whole body, pooled 8.46 NT 7001 CAGCGT Fidalgo Bay 6 Ambient 29-A
2 E03 471b 108.0 3.24 15 Larval whole body, pooled 5.2 NT 7055 ACAACG Fidalgo Bay 6 High 19-A
2 D12 472b 97.0 3.61 15 Larval whole body, pooled 2.42 NT 7052 GGTATA Fidalgo Bay 6 High 21-A
2 E06 552b 74.6 4.69 15 Larval whole body, pooled 1.84 NT 7058 ATAAGA Oyster Bay C2 6 Ambient 41-A

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Laura’s Notebook: April 23, 2020 – Oly methylation characterization

Met with Katherine and Steven a couple weeks ago and updated them on my DML, DMG, and size-associated loci (SAL) analyses. I am testing out using RMarkdown to write my results, so check out this notebook entry for a summary of these activities:

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One thing I was missing was a general characterization of O. lurida methylation patterns. This is what I tackled the past couple days. To do so, I merged methylation data from all 18 samples (18 .bam files) into one .bam files, and ran that through MethylKit for some quick summary stats, then called methylation status using 50% threshold, filtered for 5x, and annotated using the O. lurida feature files. This is all posted in my RMarkdown notebook, 01b-General-Methylation-Patterns.html.

Here are barplots showing the % of methylated loci that overlap with genome features; note that I should also include a bar showing where all CpG loci are:

image

One interesting observation from this analysis is that our filtering is excluding most of the 0% methylated loci. Check out the below % methylation frequency plots:

  1. When all samples are combined into one file, and we filter for only 2x coverage, we get a typical peak at 0% methylation. We also see a small peak at 50% methylation, probably b/c there are a lot of loci with 2x coverage, and 1 of the 2 reads was methylated.
  2. When we filter for 5x coverage (again, with all samples merged), the 0% methylation peak dramatically reduces.
  3. When filtered for 10x coverage, the 0% peak nearly disappears.

This explains why we don’t have much 0% methylation in our data – low coverage for un-methylated loci. This may mean that our analyses omits loci where methylation is substantially different between populations (i.e. 100% vs. 0% methylated). But, can we infer that low-coverage is a result of low methylation, or just poor coverage? Will dig into this further.

meth_coverage

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Laura’s Notebook: Oly OA RNA isolation – juvenile ctenidia

I’m done with my adult ctenidia & larvae libraries, and have enough kit leftover for ~17 more samples. I’ve decided to prep a few of my juvenile samples, which were collected at the end of the summer deployment. It could be very interesting to assess differences in juveniles and whether they are similar to those observed in the parents that were directly exposed.

I’m doing the whole body samples collected from the Hood Canal and Fidalgo Bay populations after they were deployed in Port Gamble Bay. I have n=4 per population and parental pH treatment (high or ambient). I had wanted to do ctenidia tissues, but then I checked out the frozen samples and noticed that they definitely are not just gill – lots of mantle tissue in there too. Therefore, I decided to do whole-body samples to try to standardize the tissue type.

Step 1: Homogenize tissue (March 6th, 2020)

Need: LN, dry ice, bleach, DI water, mortar + pestle, metal spatulas

  • Added 1mL RNAzol to 1.5 mL microcentrifuge tubes.
  • Cleaned mortars, pestles, and metal spatulas. Did this by cleaning under hot water, soaking in 10% bleach/DI solution for a minimum of 10 minutes, rinsing thoroughly with DI water, then rinsing with 190 proof ethanol and letting dry.
  • Put tubes with RNAzol on scale. Ground tissues to powder, scraped with metal spatula and carefully transferred powder to tube. Added approximately 50 mg.
  • I did 8 samples at a time (the # of mortar+pestle kits I have), then cleaned and repeated with another 8.

Step 2: RNA isolation (March 7th, 2020)

Need: RNAzol, DEPC-treated water, isopropanol, 200-proof ethanol, 1.7 mL tubes

Followed the RNAzol® RT RNA Isolation Reagent protocol for Total RNA isolation, using half of my homogenate, so 500 uL.

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Laura’s Notebook: February 2020 goals

Yikes, it’s been a few months …

  • Finish QuantSeq libraries – last step is to process some deployed juvnile Olys (RNA isolation, library prep)
  • Coordinate sequencing – UW or UMinnesota?
  • DMG and DMR analysis on Oly methylation data. Make sure that I’ve controlled for genotype (i.e. differences aren’t due to presence/absence of certain genes/loci) – does the filtering accomplish this?
  • Prepare and deliver presentation at Aquaculture America 2020
  • Revise Oly Temp/Food draft, and rough draft of introduction
  • Submit Polydora paper to Aquaculture Research
  • Meet OA/Reproduction deadlines

Also … Met with Krista – we are a go on the internship. I will get my hands on the data as soon as it’s ready (April?). She supports me doing the NSF INTERN in Fall/Winter, so should continue pursuing that. Need to have full analysis of data by November at latest.

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Laura’s Notebook: Oly DMG analysis, Jan. 30th, 2020

Today I identified 46 differentially methylated genes among two Olympia oyster populations, Hood Canal and South Sound. This was performed using a binomial GLM and Chi-square tests. The script was adapted from Hollie Putnam’s script (/hputnam/Geoduck_Meth/master/RAnalysis/Scripts/GM.Rmd), which may have been adopted from the Lieu et al. 2018 paper .

The analysis was performed in a RMarkdown notebook, please see that here: 09-DMG-analysis

Here are the GO terms associated with genes of known function. Some notes:
– 18 out of the 46 genes were annotated with GO terms
– 9 out of the 46 genes were annotated but did not have associated GO terms (may have to find those manually …)
– 19 out of the 46 genes were of unknown function

term ID description frequency pin? log10 p-value uniqueness dispensability
GO:0006468 protein phosphorylation 4.137 % -3.7877 0.40 0.00
GO:0006807 nitrogen compound metabolic process 38.744 % -2.2764 0.78 0.03
GO:0006207 ‘de novo’ pyrimidine nucleobase biosynthetic process 0.192 % -2.2764 0.46 0.06
GO:0006281 DNA repair 2.234 % -2.4853 0.50 0.20
GO:0006030 chitin metabolic process 0.077 % -1.6311 0.49 0.21
GO:0006520 cellular amino acid metabolic process 5.591 % -2.2764 0.42 0.35
GO:0006412 translation 5.686 % -2.4853 0.28 0.55
GO:0016567 protein ubiquitination 0.523 % -1.4336 0.44 0.56

image

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Laura’s Notebook: QuantSeq – inventory of finished libraries

Library prep is complete! However, I will need to redo many 😦 Based on Bioanalyzer results, any libraries with concentration <1.0 ng/uL will need to be re-done. Out of the total 132 samples that I prepped, 34 had concentrations that were LOW or <1.0 ng/uL (according to Qubit). That’s about a 25% incompletion rate. Here is the final inventory.

Next step – redo the 34 samples. Need to decide whether I should re-isolate RNA, or just try to re-do the library generation. This is a question for lab meeting tomorrow.

Ctenidia

Cohort pCO2 HOMOGENATE TUBE # TISSUE TYPE TISSUE SAMPLE # [RNA] after Dnase treatment (ng/uL) End Point PCR cycles ds cDNA concentration
Dabob Bay High 291 CTENIDIA HL10-10 158.0 15 LOW
Dabob Bay High 292 CTENIDIA HL10-11 29.6 15 1.32
Dabob Bay High 293 CTENIDIA HL10-12 39.6 16 1.03
Dabob Bay High 294 CTENIDIA HL6-10 110.0 16 1.95
Dabob Bay High 295 CTENIDIA HL6-11 34.8 18 LOW
Dabob Bay High 296 CTENIDIA HL6-12 180.0 15 3.46
Dabob Bay High 298 CTENIDIA HL6-14 182.0 15 3.08
Dabob Bay High 299 CTENIDIA HL6-15 50.4 15 2.46
Dabob Bay Ambient 301 CTENIDIA HL10-19 75.8 15 2.82
Dabob Bay Ambient 302 CTENIDIA HL10-20 62.4 14 4.90
Dabob Bay Ambient 306 CTENIDIA HL10-21 136.0 18 LOW
Dabob Bay Ambient 304 CTENIDIA HL6-19 200.0 15 2.80
Dabob Bay Ambient 305 CTENIDIA HL6-20 75.2 16 2.96
Dabob Bay Ambient 303 CTENIDIA HL6-21 95.2 14 2.40
Dabob Bay Ambient 307 CTENIDIA HL6-16 89.4 TOO LOW TOO LOW
Dabob Bay Ambient 308 CTENIDIA HL6-17 73.6 16 2.48
Dabob Bay Ambient 309 CTENIDIA HL6-18 170.0 14 1.46
Oyster Bay High 311 CTENIDIA SN6-16 158.0 16 3.76
Oyster Bay High 312 CTENIDIA SN6-17 90.6 15 1.58
Oyster Bay High 313 CTENIDIA SN6-18 72.4 15 1.81
Oyster Bay High 314 CTENIDIA SN6-19 42.2 17 LOW
Oyster Bay High 315 CTENIDIA SN6-20 148.0 15 2.32
Oyster Bay High 316 CTENIDIA SN6-21 146.0 16 3.74
Oyster Bay High 317 CTENIDIA SN6-22 158.0 15 2.14
Oyster Bay High 318 CTENIDIA SN6-23 174.0 16 4.08
Oyster Bay High 319 CTENIDIA SN6-24 77.6 16 0.64
Oyster Bay Ambient 321 CTENIDIA SN6-25 148.0 15 3.64
Oyster Bay Ambient 322 CTENIDIA SN6-26 44.6 16 NOT QUANTIFIED
Oyster Bay Ambient 323 CTENIDIA SN6-27 102.0 15 3.70
Oyster Bay Ambient 324 CTENIDIA SN6-28 172.0 16 0.78
Oyster Bay Ambient 325 CTENIDIA SN6-29 180.0 14 2.92
Oyster Bay Ambient 326 CTENIDIA SN6-30 130.0 15 4.04
Oyster Bay Ambient 327 CTENIDIA SN6-31 85.2 15 1.97
Oyster Bay Ambient 328 CTENIDIA SN6-32 156.0 14 2.12
Oyster Bay Ambient 329 CTENIDIA SN6-33 162.0 15 0.33
Fidalgo Bay High 331 CTENIDIA NF6-16 42.2 16 LOW
Fidalgo Bay High 332 CTENIDIA NF6-17 65.8 17 LOW
Fidalgo Bay High 333 CTENIDIA NF6-18 78.6 15 2.98
Fidalgo Bay High 334 CTENIDIA NF6-19 64.8 17 LOW
Fidalgo Bay High 335 CTENIDIA NF6-20 180.0 15 2.58
Fidalgo Bay High 336 CTENIDIA NF6-21 94.8 20 LOW
Fidalgo Bay High 337 CTENIDIA NF6-22 194.0 15 4.02
Fidalgo Bay High 338 CTENIDIA NF6-23 81.6 16 2.74
Fidalgo Bay High 339 CTENIDIA NF6-24 77.2 16 1.91
Fidalgo Bay Ambient 341 CTENIDIA NF6-25 89.6 14 1.31
Fidalgo Bay Ambient 342 CTENIDIA NF6-26 162.0 15 1.78
Fidalgo Bay Ambient 343 CTENIDIA NF6-27 114.0 14 2.58
Fidalgo Bay Ambient 344 CTENIDIA NF6-28 25.0 15 1.58
Fidalgo Bay Ambient 345 CTENIDIA NF6-29 190.0 14 3.50
Fidalgo Bay Ambient 346 CTENIDIA NF6-30 43.6 14 1.36
Fidalgo Bay Ambient 347 CTENIDIA NF6-31 69.0 15 LOW
Fidalgo Bay Ambient 348 CTENIDIA NF6-32 54.4 16 3.06
Fidalgo Bay Ambient 349 CTENIDIA NF6-33 82.0 15 1.90

Larvae

Spawning Tank Cohort Treatment Homo./RNA TUBE # [RNA] after Turbo Dnase treatment (ng/uL) End Point PCR cycles ds cDNA concentration
HL-10 Ambient Dabob Bay 10 Ambient 401 93.4 16 1.24
HL-10 Ambient Dabob Bay 10 Ambient 402 114.0 16 2.76
HL-10 Ambient Dabob Bay 10 Ambient 403 136.0 14 4.16
HL-10 Ambient Dabob Bay 10 Ambient 404 112.0 15 2.92
HL-10 Low Dabob Bay 10 Low 411 72.6 16 3.08
HL-10 Low Dabob Bay 10 Low 412 31.2 17 LOW
HL-10 Low Dabob Bay 10 Low 413 130.0 15 1.97
HL-10 Low Dabob Bay 10 Low 414 168.0 15 0.86
HL-6 Ambient Dabob Bay 6 Ambient 421 57.6 14 1.63
HL-6 Low Dabob Bay 6 Low 431b 83.0 16 3.36
HL-6 Low Dabob Bay 6 Low 432 74.0 17 LOW
HL-6 Low Dabob Bay 6 Low 434 63.0 15 5.58
NF-10 Ambient A Fidalgo Bay 10 Ambient 441 16.2 15 LOW
NF-10 Ambient A Fidalgo Bay 10 Ambient 442b 69.8 17 LOW
NF-10 Ambient A Fidalgo Bay 10 Ambient 443 60.2 15 1.36
NF-10 Ambient B Fidalgo Bay 10 Ambient 444 70.6 14 4.08
NF-10 Ambient B Fidalgo Bay 10 Ambient 445 160.0 15 4.00
NF-10 Low B Fidalgo Bay 10 Low 451 68.4 14 2.24
NF-10 Low A Fidalgo Bay 10 Low 452b 97.2 16 2.68
NF-10 Low B Fidalgo Bay 10 Low 453 196.0 14 0.97
NF-6 Ambient B Fidalgo Bay 6 Ambient 461b 84.0 14 2.02
NF-6 Ambient A Fidalgo Bay 6 Ambient 462b 106.0 16 0.21
NF-6 Low B Fidalgo Bay 6 Low 471b 108.0 15 5.20
NF-6 Low B Fidalgo Bay 6 Low 472b 97.0 15 2.42
NF-6 Low B Fidalgo Bay 6 Low 473 124.0 14 2.34
NF-6 Low A Fidalgo Bay 6 Low 474 77.2 14 3.02
NF-6 Low A Fidalgo Bay 6 Low 475 27.4 14 2.44
NF-6 Low A Fidalgo Bay 6 Low 476 39.2 14 4.24
NF-6 Low A Fidalgo Bay 6 Low 477 164.0 15 2.28
SN-10 Ambient A Oyster Bay C1 10 Ambient 481 89.2 14 0.88
SN-10 Ambient B Oyster Bay C1 10 Ambient 482 22.2 14 1.42
SN-10 Ambient B Oyster Bay C1 10 Ambient 483 99.0 16 1.22
SN-10 Ambient A Oyster Bay C1 10 Ambient 484 58.4 15 1.67
SN-10 Ambient B Oyster Bay C1 10 Ambient 485 19.1 14 2.90
SN-10 Ambient A Oyster Bay C1 10 Ambient 486b 148.0 16 LOW
SN-10 Ambient B Oyster Bay C1 10 Ambient 487 118.0 16 1.27
SN-10 Ambient A Oyster Bay C1 10 Ambient 488 60.0 14 3.06
SN-10 Ambient A Oyster Bay C1 10 Ambient 489 68.0 15 3.84
SN-10 Ambient B Oyster Bay C1 10 Ambient 490 186.0 14 2.32
SN-10 Ambient B Oyster Bay C1 10 Ambient 491 58.4 15 6.64
SN-10 Ambient B Oyster Bay C1 10 Ambient 492 82.6 15 2.02
SN-10 Low B Oyster Bay C1 10 Low 41 108.0 17 LOW
SN-10 Low B Oyster Bay C1 10 Low 43 156.0 15 2.48
SN-10 Low B Oyster Bay C1 10 Low 46 98.6 18 LOW
SN-10 Low B Oyster Bay C1 10 Low 47 128.0 17 LOW
SN-10 Low A Oyster Bay C1 10 Low 44 41.4 TOO LOW TOO LOW
SN-10 Low B Oyster Bay C1 10 Low 506 29.2 15 1.63
SN-10 Low A Oyster Bay C1 10 Low 45 25.1 20 LOW
SN-6 Ambient B Oyster Bay C1 6 Ambient 35 94.4 20 LOW
SN-6 Ambient B Oyster Bay C1 6 Ambient 513 142.0 15 5.92
SN-6 Ambient B Oyster Bay C1 6 Ambient 39 89.8 15 2.04
SN-6 Ambient B Oyster Bay C1 6 Ambient 37 74.6 20 LOW
SN-6 Ambient A Oyster Bay C1 6 Ambient 34 104.0 18 LOW
SN-6 Low A Oyster Bay C1 6 Low 521 66.6 17 LOW
SN-6 Low A Oyster Bay C1 6 Low 522 32.2 16 2.42
SN-6 Low B Oyster Bay C1 6 Low 523 71.4 16 2.16
SN-6 Low B Oyster Bay C1 6 Low 524 63.0 14 3.24
SN-6 Low B Oyster Bay C1 6 Low 525 140.0 14 2.44
SN-6 Low A Oyster Bay C1 6 Low 526 138.0 14 2.20
SN-6 Low B Oyster Bay C1 6 Low 527 124.0 15 1.42
SN-6 Low B Oyster Bay C1 6 Low 528 87.6 14 4.48
SN-6 low A Oyster Bay C1 6 Low 529 56.8 15 2.86
K-10 Ambient Oyster Bay C2 10 Ambient 531 95.4 15 2.10
K-10 Ambient Oyster Bay C2 10 Ambient 532 93.6 14 2.12
K-10 Ambient Oyster Bay C2 10 Ambient 533 6.5 17 LOW
K-10 Low Oyster Bay C2 10 Low 541 44.4 15 1.40
K-10 Low Oyster Bay C2 10 Low 542 32.8 16 2.22
K-10 Low Oyster Bay C2 10 Low 543 162.0 14 5.24
K-6 Ambient Oyster Bay C2 6 Ambient 551 96.4 14 1.69
K-6 Ambient Oyster Bay C2 6 Ambient 552b 74.6 15 1.84
K-6 Ambient Oyster Bay C2 6 Ambient 553 186.0 14 3.28
K-6 Ambient Oyster Bay C2 6 Ambient 554 188.0 14 2.38
K-6 Low Oyster Bay C2 6 Low 561 28.0 17 LOW
K-6 Low Oyster Bay C2 6 Low 562 126.0 17 LOW
K-6 Low Oyster Bay C2 6 Low 563 47.0 15 0.19
K-6 Low Oyster Bay C2 6 Low 564 152.0 14 2.24
K-6 Low Oyster Bay C2 6 Low 565 31.2 17 LOW
homog. Control RNA Control 571 LOW 18 LOW
homog. Control RNA Control 572 TOO LOW TOO LOW

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Laura’s Notebook: QuantSeq library amplification, purification, and QA

The final step in the QuantSeq library prep is to amplify my cDNA libraries (using the optimal cycle number) and then purify. Finally, to assess quantity and quality of finished libraries I use the Qubit High Sensitivity DNA kit to measure cDNA concentration, and the Bioanalyzer High Sensitivity DNA chip kit to measure fragment lengths. I worked in batches based on the number of cycles needed to amplify.

IMPORTANT NOTE: I have 2 sets of indexes with the same numbers. I therefore prepped the ctenidia samples with one set, and the larvae with another set. Therefore, I SHOULD NOT run ctenidia and larval samples in the same lane (since two samples have the same index number).

The spreadsheet where I have all this information organized is in the laura-quantseq repo, file is: 2019-July_RNA-isolation-ctenidia-larvae.xlsx

12/30/2019 – 16 cycles

  • I noticed that I didn’t have the full 17 uL in any of the ds cDNA libraries, probably missing ~ 2uL. Not sure why, possibly due to pipette loss?
  • Sample #331 volume depleted after PCR, maybe due to evaporation b/c there was a small crease in the foil? This was located at well A1 – should ensure tight seal for all future plates.

Amplification plate configuration

Snip20200106_8

1/1/2020 – 14 cycles

  • Again, samples <17 uL.

Amplification plate configuration

Snip20200106_9

1/2/2020 – 15 cycles

  • Again, most samples <17 uL.

Amplification plate configuration

Snip20200106_10

1/4/2020 – 17, 18 and 20 cycles

  • I amplified 3 batches of samples today – those needing 17, 18 and 20 cycles (ran in that order). I held amplified samples at 10C until all groups were done, then combined all samples onto one plate, purified, and quantified.
  • Unfortunately, none of these libraries amplified! I don’t know whether there was an error in the amplification step, OR if the high number of cycle needed represent low concentration/quality. Looking/thinking back through all my steps I do not see any possible errors that could have been made in the PCR mix (it’ a simple master mix using 2 reagents), or during purification. Regardless, I will need to re-do all these libraries!

Amplification plate configuration & index

Snip20200106_11

Snip20200106_12

Snip20200106_13

Amplified and Indexed QuantSeq cDNA libraries

This is the plate configuration of the finished libraries. Based on the quantification/Bioanalyzer results, some of these libraries will need to be re-done. However, this is an important map!

Snip20200106_14

Snip20200106_15

Bioanalyzer quality check

  • I ran 1-2 chips per amplification group. The first 2 chips, which I did for the 16 cycle group, worked great! Results below.
  • I had some issues with the next few chips. Not sure why, but I suspect that it had to do with the fact that I used the Seeb lab’s pipette/pipette tips.
  • I have other chip results, but did not take screen shots at the time – will follow up with those, but for the time being all files are saved in the repo: laura-quantseq/data/library-prep

Results for samples ran for 16 cycles

imageimageSnip20200106_6image

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Laura’s Notebook: QuantSeq Library Generation Batches 3 and 4

IMG_9282

IMG_9283

Batch 3 Sample Information

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 35 94.4 3.71 1.29 350
2 524 63.0 5.00 315
3 453 196.0 1.79 3.21 350
4 554 188.0 1.86 3.14 350
5 442b 69.8 5.00 349
6 489 68.0 5.00 340
7 462b 106.0 3.30 1.70 350
8 533 6.5 5.00 32.6
9 522 32.2 5.00 161
10 474 77.2 4.53 0.47 350
11 452b 97.2 3.60 1.40 350
12 443 60.2 5.00 301
13 477 164.0 2.13 2.87 350
14 526 138.0 2.54 2.46 350
15 562 126.0 2.78 2.22 350
16 432 74.0 4.73 0.27 350
17 37 74.6 4.69 0.31 350
18 413 130.0 2.69 2.31 350
19 45 25.1 5.00 125.5
20 561 28.0 5.00 140
21 542 32.8 5.00 164
22 527 124.0 2.82 2.18 350
23 492 82.6 4.24 0.76 350
24 475 27.4 5.00 137
25 541 44.4 5.00 222
26 565 31.2 5.00 156
27 B3 – NTC1 NA 5.00 #VALUE!
28 B3 – NTC2 NA 5.00 #VALUE!

Batch 3 Plate Configuration

1 2 3 4 5 6 7 8
A 35 524 453 554 442b 489 462b
B
C 533 522 474 452b 443 477 526
D
E 562 432 37 413 45 561 542
F
G 527 492 475 541 565 B3 – NTC1 B3 – NTC2
H

Batch 4 Sample Information

Sample order in plate Sample No. [RNA] (ng/ul) Vol RNA used Vol H2O to add ng RNA used
1 571 LOW 5.00 NA
2 525 140.0 2.50 2.50 350
3 563 47.0 5.00 235
4 404 112.0 3.13 1.88 350
5 484 58.4 5.00 292
6 531 95.4 3.67 1.33 350
7 34 104.0 3.37 1.63 350
8 490 186.0 1.88 3.12 350
9 523 71.4 4.90 0.10 350
10 473 124.0 2.82 2.18 350
11 485 19.1 5.00 95.5
12 402 114.0 3.07 1.93 350
13 487 118.0 2.97 2.03 350
14 476 39.2 5.00 196
15 421 57.6 5.00 288
16 553 186.0 1.88 3.12 350
17 41 108.0 3.24 1.76 350
18 46 98.6 3.55 1.45 350
19 551 96.4 3.63 1.37 350
20 486b 148.0 2.36 2.64 350
21 445 160.0 2.19 2.81 350
22 43 156.0 2.24 2.76 350
23 506 29.2 5.00 146
24 482 22.2 5.00 111
25 412 31.2 5.00 156
26 488 60.0 5.00 300
27 47 128.0 2.73 2.27 350
28 B4 – NTC2 NA 5.00 0

Batch 4 Plate Configuration

1 2 3 4 5 6 7 8
A 571 525 563 404 484 531 34
B
C 490 523 473 485 402 487 476
D
E 421 553 41 46 551 486b 445
F
G 43 506 482 412 488 47 B4 – NTC2
H

Notes

  • Aliquoted 20 uL RNA into new tubes and froze in -80. Except, for the following samples I aliquoted 7 uL: 34, 35, 41, 46, 46
  • Sample #35 was not fully sealed during either the RNA removal step or the second strand synthesis step, and I believe I lost some volume to evaporation. I proceeded normally, but will see how that library looks.
  • Not sure if I mentioned this previously, but I’m using 350 ng RNA, except for low concentration samples (I use 5 uL to get the maximum amount of RNA possible).
  • To “quickly spin down” samples at room temperature I put the sample plate in the centrifuge, push start, then push stop immediately when the speed does not read 0. I also set the temperature to room temperature so it doesn’t actively cool.

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