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.

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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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