|
| Status |
Public on Mar 31, 2022 |
| Title |
seedpod-pooled Iso-seq |
| Sample type |
SRA |
| |
|
| Source name |
seedpod
|
| Organism |
Medicago sativa |
| Characteristics |
tissue: seedpod
developmental stage: 20 week days old plant
genotype: RegenSY27x
|
| Growth protocol |
RegenSY27x alfalfa plants were grown at 22oC and 18oC day/night temperatures with a photoperiod of 16 hours and three replicates of 8-week-old leaf, stem, flower, seedpod, root and nodule tissue harvested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was extracted from these plants using a Qiagen RNeasy Plant Mini kit according to the manufacturer's instructions, pooled and used to construct libraries
The libraries were validated using a High Sensitivity Chip on the Agilent 2100 Bioanalyzer.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Sequel |
| |
|
| Data processing |
Library strategy: Iso-seq
PacBio® Iso-seq analysis, the same RNA was prepared according to the Iso-Seq Sample Preparation Procedure
The data processed using tools from the PacBio SMRT Link v9.0 distribution, beginning with conversion of subreads into consensus sequences using ccs v4.2.0 with default parameters.
Consensus reads used in subsequent analyses resulted from removal of primer sequences using lima (v1.11.0, --isoseq option with NEB primers) and identification of full length non-concatamer (flnc) sequences with polyA tails trimmed using isoseq3 refine (v3.3.0) with default parameters
The resulting flnc sequences were aligned to the Medicago truncatula A17 v4.0 reference genome (medtr.A17_HM341.gnm4) using minimap2 (2.17-r954-dirty, with options --splice --cs ) and the subsets of reads aligning to chr2:3696725-3700846 and chr4:35437929-35444569 were respectively taken as MsPHO2-B haplotypes corresponding to Medtr2g013650/ MtrunA17_Chr2g0282211, and MsPHO2-C haplotypes corresponding to Medtr4g088835/ MtrunA17Chr4g0047301 (MsPHO2-2)
The set of reads corresponding to each of the two loci were independently processed for possible allele-defining variation using samtools (v1.10) mpileup with options --excl-flags 3844 --output-QNAME, and the haplotype-defining variation was assessed using script phase_mpileup.pl (available from https://github.com/adf-ncgr/haploallele_utils/releases/tag/miller_et_al_2021) using options --max_hap_dist=6000 --ploidy=4 (and including --min_hap_coverage=4 for PHO2-C); the strategy used in the script was loosely based on the isophase strategy described in (WANG et al. 2020).
|
| |
|
| Submission date |
Feb 25, 2022 |
| Last update date |
Mar 31, 2022 |
| Contact name |
Shaun J J Curtin |
| E-mail(s) |
shaun.curtin@usda.gov, curti242@umn.edu
|
| Phone |
6126255715
|
| Organization name |
USDA-ARS-PSRU
|
| Department |
Agronomy and Plant Genetics
|
| Street address |
1991 Upper Buford Circle, 411 Borlaug Hall
|
| City |
St Paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
| |
|
| Platform ID |
GPL31996 |
| Series (2) |
| GSE197480 |
Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate [Iso-seq] |
| GSE197482 |
Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate |
|
| Relations |
| BioSample |
SAMN26264205 |
| SRA |
SRX14293372 |
