 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 01, 2019 |
| Title |
Env sCD4 Sorted Library A; Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
Synthetic coding sequences
|
| Organism |
Human immunodeficiency virus 1 |
| Characteristics |
protein: Env (gp140)
sort gate: Top 0.3% of population for binding signal (10 nM sCD4(D1-D2))
# collected cells: 115,000
|
| Treatment protocol |
Cells expressing DNA libraries of HIV-1(DU422) gp140 mutants (cloned into pCEP4, Invitrogen) were FACS sorted for binding to soluble CD4 (domains D1-D2) or broadly-neutralizing antibody PG16. The extracellular region of Env (gp140) was fused at its C-terminus vi a gly/ser-rich linker and 6H-tag to a canonical transmembrane helix for surface display. Three single site-saturation mutagenesis libraries were created, spanning residues 31-279 (Library A; numbering based on the HXB2 reference strain), 280-577 (Library B), and 578-677 (Library C).
|
| Growth protocol |
Expi293F cells cultured in Expi293 Expression Medium (Life Technologies)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested from sorted cells using GeneJet RNA Purification Kit (Thermo Scientific) and cDNA prepared using Accuscript Hi-Fi (Agilent Genomics, Libraries A, B and C) or SuperScript IV VILO (Invitrogen, Library A) primed with: random hexamers and oligo-dT (Library A, SuperScript), DU422gp160_libA_RT_rev2 (GGCCTAGTGCATTTAATCTC, Library A, Accuscript), DU422gp160_libB_RT_rev2 (GTTGCTGGTCCTTAAGATAC, Library B), and EBV_reverse (GTGGTTTGTCCAAACTCATC, Library C).
In the first round of PCR, the gp140 coding sequences were amplified as overlapping fragments of ~800 bp each.
Primer pair (Library A Fragment 1): HiSeq_DU422gp160_52_F (TCTTTCCCTACACGACGCTCTTCCGATCTTGTAGCGTCAGTGCTTGC) and HiSeq_DU422gp160_624_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACACTTCAGTATGGCGTACC)
Primer pair (Library A Fragment 2): HiSeq_DU422gp160_279_F (TCTTTCCCTACACGACGCTCTTCCGATCTCATGTGGAAGAACGACATGG) and HiSeq_DU422gp160_850_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGGACTTGTTGAGATGGAC)
Primer pair (Library B Fragment 1): HiSeq_DU422gp160_808_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCGAAGTGAGAACTTGACGA) and HiSeq_DU422gp160_1489_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCAACCGCTCGCTTTTC)
Primer pair (Library B Fragment 2): HiSeq_DU422gp160_1035_F (TCTTTCCCTACACGACGCTCTTCCGATCTAACTGCGGGAGCATTACAAC) and HiSeq_DU422gp160_1713_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGATACCGCTCAATCGCGA)
Primer pair (Library C): HiSeq_DU422gp160_1671_F (TCTTTCCCTACACGACGCTCTTCCGATCTGGGGCATAAAGCAACTG) and HiSeq_6His-Linker_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGATGGTGATGATGTCC)
In a second round of PCR, Illumina adaptors and experiment-specific barcodes were added.
Primer pair for adding I5 and I7 Illumina adaptors: MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced on a HiSeq 2500 using a 2x250 nt paired end protocol, though data is analyzed as single reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Deep mutational scan of DU422 gp140 for binding to soluble CD4
|
| Data processing |
Data was analyzed using Enrich: http://depts.washington.edu/sfields/software/enrich/
Fuser script: FragmentA1 read 1: python Paired_read_fuser.py --path fragA1_R1/ --read1 DU422gp140_A_R1.fastq.bz2 --read2 DU422gp140_A_R1.fastq.bz2 --wtseq AATCTCGACCTTTGGGTCACCGTATACTACGGAGTCCCCGTGTGGAAAGAGGCTAAGACGACCCTGTTTTGTGCGTCAGACGCGAAAGCGTATGACAAGGAAGTGCATAACGTGTGGGCAACACACGCATGTGTTCCGACGGACCCTAACCCGCAGGAAATCGTCCTTGAAAACGTGACCGAAAACTTCAACATGTGGAAGAACGACATGGTTGACCAA --read1_overlap_start 19 --read1_overlap_end 238 --mode R1
Fuser script: Fragment A1 read 2: python Paired_read_fuser.py --path fragA1_R2/ --read1 DU422gp140_A_R2.fastq.bz2 --read2 DU422gp140_A_R2.fastq.bz2 --wtseq TCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAAAAAGTCTACGCCCTTTTCTATAAACCGGATGTCGTGCCACTTAACGGTGGTGAGCACAATGAGACAGGTGAGTACATTCTCATCAACTGCAATTCATCTACAATAACGCAAGCATGTCCGAAAGTTTCCTTCGATCCGATCCCTATTCACTACTGCGCTCCTGCA --read2_overlap_start 25 --read2_overlap_end 229 --mode R2
Fuser script: Fragment A2 read 1: python Paired_read_fuser.py --path fragA2_R1/ --read1 DU422gp140_A_R1.fastq.bz2 --read2 DU422gp140_A_R1.fastq.bz2 --wtseq GACCAAATGCACGAGGACATAATCAGTCTTTGGGATCAATCCTTGAAGCCCTGTGTGAAGCTGACGCCTTTGTGCGTAACTTTGAACTGTAAAAACGTAAATATTTCAGCGAATGCTAACGCGACCGCTACACTTAATTCAAGTATGAACGGCGAAATAAAGAATTGCTCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAA --read1_overlap_start 22 --read1_overlap_end 229 --mode R1
Fuser script: Fragment A2 read 2: python Paired_read_fuser.py --path fragA2_R2/ --read1 DU422gp140_A_R2.fastq.bz2 --read2 DU422gp140_A_R2.fastq.bz2 --wtseq GCAGGGTACGCCATACTGAAGTGTAACAATAAAACGTTTAACGGAACAGGCCCTTGCAATAATGTAAGTACTGTCCAGTGCACGCATGGCATAAAACCCGTCGTGAGCACACAACTTCTTCTTAACGGATCACTCGCCGAGGAAGAGATCATCGTGCGAAGTGAGAACTTGACGAAT --read2_overlap_start 35 --read2_overlap_end 212 --mode R2
Fuser script: Fragment B1 read 1: python Paired_read_fuser.py --path fragB1_R1/ --read1 DU422gp140_B_R1.fastq.bz2 --read2 DU422gp140_B_R1.fastq.bz2 --wtseq AATATAAAGACGATCATAGTCCATCTCAACAAGTCCGTCGAGATTAAATGCACTAGGCCCAACAACAATACGCGGAAATCCGTACGGATAGGTCCTGGACAAACATTCTATGCTACCGGTGAGATTATAGGTGATATACGCGAAGCTCATTGCAATATCAGTAGGGAGACCTGGAATTCCACTTTGATCCAAGTGAAAGAAAAACTGCGGGAGCATTACAAC --read1_overlap_start 22 --read1_overlap_end 244 --mode R1
Fuser script: Fragment B1 read 2: python Paired_read_fuser.py --path fragB1_R2/ --read1 DU422gp140_B_R2.fastq.bz2 --read2 DU422gp140_B_R2.fastq.bz2 --wtseq CCGCCGATTGAAGGGAATATTACTTGCAAGAGCAACATAACTGGTCTTCTTCTCACATGGGATGGTGGAGAGAACAGTACCGAAGGCGTTTTTAGGCCTGGCGGTGGCAATATGAAGGATAACTGGCGCTCAGAACTCTATAAGTACAAAGTCGTCGAAATAAAGCCCTTGGGCGTTGCGCCTACGAAAAGTAAACGAAAAGTAGTAGGGAGG --read2_overlap_start 18 --read2_overlap_end 231 --mode R2
Fuser script: Fragment B2 read 1: python Paired_read_fuser.py --path fragB2_R1/ --read1 DU422gp140_B_R1.fastq.bz2 --read2 DU422gp140_B_R1.fastq.bz2 --wtseq AAAACGATCAAATTTGAACCTTCTTCTGGAGGTGACCTGGAGGTTACGACGCACAGTTTTAACTGTAGGGGAGAATTCTTCTACTGCGATACGACCAAGCTGTTCAACGAGACTAAGCTGTTTAACGAGTCAGAATACGTTGACAATAAGACTATTATCTTGCCCTGCCGGATAAAGCAAATTATCAATATGTGGCAAGAAGTGGGAAGGGCAATGTACGCG --read1_overlap_start 20 --read1_overlap_end 242 --mode R1
Fuser script: Fragment B2 read 2: python Paired_read_fuser.py --path fragB2_R2/ --read1 DU422gp140_B_R2.fastq.bz2 --read2 DU422gp140_B_R2.fastq.bz2 --wtseq GAAAAGCGAGCGGTTGGCTTGGGTGCAGTGCTTCTCGGGTTCCTGGGAGCTGCGGGGTCAACAATGGGAGCTGCTTCCATAACCTTGACTGTGCAGGCAAGGCAGCTGCTGAGTGGGATAGTGCAGCAACAATCCAATCTGCTTAGGGCAATTGAAGCTCAGCAACATCTTTTGCAGCTCACAGTCTGGGGCATAAAGCAACTGCAA --read2_overlap_start 13 --read2_overlap_end 220 --mode R2
Fuser script: Fragment C read 1: python Paired_read_fuser.py --path fragC_R1/ --read1 DU422gp140_C_R1.fastq.bz2 --read2 DU422gp140_C_R1.fastq.bz2 --wtseq ACCAGAGTCCTCGCGATTGAGCGGTATCTTAAGGACCAGCAACTGCTGGGACTCTGGGGGTGTTCCGGGAAGCTTATTTGTGCCACTGCGGTACCATGGAACTCCTCTTGGTCCAACAAGTCTCTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTT --read1_overlap_start 20 --read1_overlap_end 224 --mode R1
Fuser script: Fragment C read 2: python Paired_read_fuser.py --path fragC_R2/ --read1 DU422gp140_C_R2.fastq.bz2 --read2 DU422gp140_C_R2.fastq.bz2 --wtseq CTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTTCTCGAAGACAGTCAGAATCAACAAGAGAAGAATGAGAAGGATCTGTTGGCCCTTGACAGCTGGAAAAATCTTTGGAATTGGTTTGACATTACTAAC --read2_overlap_start 42 --read2_overlap_end 219 --mode R2
Aligner script: Fragment A1 read 1: python Fused_read_aligner.py --path fragA1_R1/ --infile DU422gp140_A_R1.fast_R1_qc1 --referenceDNA AATCTCGACCTTTGGGTCACCGTATACTACGGAGTCCCCGTGTGGAAAGAGGCTAAGACGACCCTGTTTTGTGCGTCAGACGCGAAAGCGTATGACAAGGAAGTGCATAACGTGTGGGCAACACACGCATGTGTTCCGACGGACCCTAACCCGCAGGAAATCGTCCTTGAAAACGTGACCGAAAACTTCAACATGTGGAAGAACGACATGGTTGACCAA --referenceAA NLDLWVTVYYGVPVWKEAKTTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKNDMVDQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment A1 read 2: python Fused_read_aligner.py --path fragA1_R2/ --infile DU422gp140_A_R2.fast_R2_qc1 --referenceDNA TCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAAAAAGTCTACGCCCTTTTCTATAAACCGGATGTCGTGCCACTTAACGGTGGTGAGCACAATGAGACAGGTGAGTACATTCTCATCAACTGCAATTCATCTACAATAACGCAAGCATGTCCGAAAGTTTCCTTCGATCCGATCCCTATTCACTACTGCGCTCCTGCA --referenceAA SFNTTTELRDKKQKVYALFYKPDVVPLNGGEHNETGEYILINCNSSTITQACPKVSFDPIPIHYCAPA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment A2 read 1: python Fused_read_aligner.py --path fragA2_R1/ --infile DU422gp140_A_R1.fast_R1_qc1 --referenceDNA GACCAAATGCACGAGGACATAATCAGTCTTTGGGATCAATCCTTGAAGCCCTGTGTGAAGCTGACGCCTTTGTGCGTAACTTTGAACTGTAAAAACGTAAATATTTCAGCGAATGCTAACGCGACCGCTACACTTAATTCAAGTATGAACGGCGAAATAAAGAATTGCTCCTTCAACACCACGACAGAACTTAGAGATAAAAAGCAA --referenceAA DQMHEDIISLWDQSLKPCVKLTPLCVTLNCKNVNISANANATATLNSSMNGEIKNCSFNTTTELRDKKQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment A2 read 2: python Fused_read_aligner.py --path fragA2_R2/ --infile DU422gp140_A_R2.fast_R2_qc1 --referenceDNA GCAGGGTACGCCATACTGAAGTGTAACAATAAAACGTTTAACGGAACAGGCCCTTGCAATAATGTAAGTACTGTCCAGTGCACGCATGGCATAAAACCCGTCGTGAGCACACAACTTCTTCTTAACGGATCACTCGCCGAGGAAGAGATCATCGTGCGAAGTGAGAACTTGACGAAT --referenceAA AGYAILKCNNKTFNGTGPCNNVSTVQCTHGIKPVVSTQLLLNGSLAEEEIIVRSENLTN --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment B1 read 1: python Fused_read_aligner.py --path fragB1_R1/ --infile DU422gp140_B_R1.fast_R1_qc1 --referenceDNA AATATAAAGACGATCATAGTCCATCTCAACAAGTCCGTCGAGATTAAATGCACTAGGCCCAACAACAATACGCGGAAATCCGTACGGATAGGTCCTGGACAAACATTCTATGCTACCGGTGAGATTATAGGTGATATACGCGAAGCTCATTGCAATATCAGTAGGGAGACCTGGAATTCCACTTTGATCCAAGTGAAAGAAAAACTGCGGGAGCATTACAAC --referenceAA NIKTIIVHLNKSVEIKCTRPNNNTRKSVRIGPGQTFYATGEIIGDIREAHCNISRETWNSTLIQVKEKLREHYN --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment B1 read 2: python Fused_read_aligner.py --path fragB1_R2/ --infile DU422gp140_B_R2.fast_R2_qc1 --referenceDNA CCGCCGATTGAAGGGAATATTACTTGCAAGAGCAACATAACTGGTCTTCTTCTCACATGGGATGGTGGAGAGAACAGTACCGAAGGCGTTTTTAGGCCTGGCGGTGGCAATATGAAGGATAACTGGCGCTCAGAACTCTATAAGTACAAAGTCGTCGAAATAAAGCCCTTGGGCGTTGCGCCTACGAAAAGTAAACGAAAAGTAGTAGGGAGG --referenceAA PPIEGNITCKSNITGLLLTWDGGENSTEGVFRPGGGNMKDNWRSELYKYKVVEIKPLGVAPTKSKRKVVGR --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment B2 read 1: python Fused_read_aligner.py --path fragB2_R1/ --infile DU422gp140_B_R1.fast_R1_qc1 --referenceDNA AAAACGATCAAATTTGAACCTTCTTCTGGAGGTGACCTGGAGGTTACGACGCACAGTTTTAACTGTAGGGGAGAATTCTTCTACTGCGATACGACCAAGCTGTTCAACGAGACTAAGCTGTTTAACGAGTCAGAATACGTTGACAATAAGACTATTATCTTGCCCTGCCGGATAAAGCAAATTATCAATATGTGGCAAGAAGTGGGAAGGGCAATGTACGCG --referenceAA KTIKFEPSSGGDLEVTTHSFNCRGEFFYCDTTKLFNETKLFNESEYVDNKTIILPCRIKQIINMWQEVGRAMYA --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment B2 read 2: python Fused_read_aligner.py --path fragB2_R2/ --infile DU422gp140_B_R2.fast_R2_qc1 --referenceDNA GAAAAGCGAGCGGTTGGCTTGGGTGCAGTGCTTCTCGGGTTCCTGGGAGCTGCGGGGTCAACAATGGGAGCTGCTTCCATAACCTTGACTGTGCAGGCAAGGCAGCTGCTGAGTGGGATAGTGCAGCAACAATCCAATCTGCTTAGGGCAATTGAAGCTCAGCAACATCTTTTGCAGCTCACAGTCTGGGGCATAAAGCAACTGCAA --referenceAA EKRAVGLGAVLLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQ --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
Aligner script: Fragment C read 1: python Fused_read_aligner.py --path fragC_R1/ --infile DU422gp140_C_R1.fast_R1_qc1 --referenceDNA ACCAGAGTCCTCGCGATTGAGCGGTATCTTAAGGACCAGCAACTGCTGGGACTCTGGGGGTGTTCCGGGAAGCTTATTTGTGCCACTGCGGTACCATGGAACTCCTCTTGGTCCAACAAGTCTCTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTT --referenceAA TRVLAIERYLKDQQLLGLWGCSGKLICATAVPWNSSWSNKSLGDIWDNMTWMQWDREISNYTNTIFRL --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R1
Aligner script: Fragment C read 2: python Fused_read_aligner.py --path fragC_R2/ --infile DU422gp140_C_R2.fast_R2_qc1 --referenceDNA CTGGGCGATATTTGGGACAATATGACGTGGATGCAGTGGGATCGCGAGATATCCAACTACACCAACACGATCTTCCGGCTTCTCGAAGACAGTCAGAATCAACAAGAGAAGAATGAGAAGGATCTGTTGGCCCTTGACAGCTGGAAAAATCTTTGGAATTGGTTTGACATTACTAAC --referenceAA LGDIWDNMTWMQWDREISNYTNTIFRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDITN --gap_max 8 --unresolvable_max 2 --maxmutrun 3 --avg_quality 25 --chaste 1 --Ncount_max 3 --use_N 0 --mode R2
MapCounts script: Example: python mapCounts.py --path directory_fragA1_R1/ --infile DU422gp140_A_R1.fast_R1_qc1_PRO_qc2
MapRatios script: Example: python mapRatios.py --path ratios_directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts script: Example: python mapParts.py --path ratios_directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: Example: python mapUnlink.py --path ratios/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size ##
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Data was then assembled from the different fragments/reads to span the full sequence in Excel. Where there was overlap, real enrichment ratios were averaged and converted back to log(base2). Maximum depletion was set to log(base2) enrichment ratio = -4.
Genome_build: Not applicable
Supplementary_files_format_and_content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution. Also includes the frequency of each mutation in the naïve plasmid library and various controls.
|
| |
|
| Submission date |
Jul 18, 2018 |
| Last update date |
Feb 01, 2019 |
| Contact name |
Erik Procko |
| E-mail(s) |
procko@illinois.edu
|
| Phone |
217-300-1454
|
| Organization name |
University of Illinois
|
| Department |
Biochemistry
|
| Lab |
RAL 318G
|
| Street address |
601 S Goodwin Ave
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platform ID |
GPL22068 |
| Series (1) |
| GSE117328 |
Deep mutational scanning of Env from the HIV-1 strain DU422 for binding to CD4 and PG16 |
|
| Relations |
| BioSample |
SAMN09689957 |
| SRA |
SRX4404912 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
