진핵 전사체 시퀀싱

기술 사양

TECHNICAL SPECIFICATIONS

Item	Details
Library Prep	Oligo(dT) bead-based mRNA enrichment
Read Alignment	STAR aligns cleaned reads to the reference genome; sequencing and library quality assessed by mapping rate, insert size and coverage uniformity
Quantification	Gene/transcript quantification against the annotation, outputting Count, FPKM, TPM and log2TPM
DE Thresholds	Read-count normalization (depth correction) → statistical modeling for P values → BH adjustment (padj); default \|log2(FoldChange)\| > 1 and padj < 0.05
Standard Delivery	Raw data + analysis report

분석 프로세스

ANALYSIS WORKFLOW

데이터 QC

어댑터 트리밍 · rRNA 평가

리드 정렬

STAR · 참조 유전체

정량

Count / FPKM / TPM

발현 QC

상관성 · PCA

차등 분석

볼케이노 · 클러스터링

GO 농축

BP / CC / MF

KEGG 농축

경로 · 버블 플롯

GSEA

GO / KEGG 유전자 세트

생물정보 분석

BIOINFORMATIC MODULES

Module	Core Methods & Metrics	Key Outputs
Sequencing Statistics	Base-quality and content distributions of raw fastq (R1/R2); 3′/5′ adapter trimming; filtering reads with mean quality < 20 or length < 50 bp and reads containing N; rRNA contamination rate assessment	Raw and cleaned data statistics tables, base-quality and content distribution plots
Alignment QC	STAR aligns cleaned reads to the reference genome; sequencing and library quality assessed at checkpoints including mapping rate, insert size and coverage uniformity	Mapping-rate statistics tables, per-sample alignment detail plots
mRNA Quantification	Gene/transcript quantification against the annotation with Count, FPKM, TPM and log2TPM outputs; Top-10 highly expressed transcripts summarized	Expression matrices, Top-10 expression tables
Expression Distribution & QC	Expression density distributions; per-sample violin/box plots; inter-sample Pearson correlation heatmaps (ideal within-group R² > 0.9) and PCA	Density plots, violin/box plots, correlation heatmaps, PCA plots
Differential Analysis	Read-count normalization (depth correction) → statistical modeling for P values → BH multiple-testing adjustment (padj); default \|log2(FoldChange)\| > 1 and padj < 0.05	Differential analysis result tables, volcano plots
DE Patterns	Expression-pattern clustering of differential genes; shared and unique DEG statistics across multiple comparisons	Pattern-clustering heatmaps, shared-DEG diagrams
GO Functional Enrichment	GO enrichment of DEGs (BP / CC / MF), reporting GeneRatio / BgRatio, enrichment factor and adjusted p-values	GO enrichment bubble and bar plots
KEGG & GSEA	KEGG pathway enrichment of DEGs; GSEA based on GO and KEGG gene sets, reporting ES, NES and adjusted p-values	KEGG enrichment plots, GSEA enrichment plots and dot plots

결과 예시

SELECTED FIGURES FROM ACTUAL REPORTS

유전자 발현 밀도

시료별 log2(TPM) 밀도 곡선으로, 발현 분포의 일관성을 평가합니다.

발현 바이올린 / 박스 플롯

시료별 유전자 발현값의 분포와 확률 밀도를 보여주며, 박스는 중앙값과 사분위수를 나타냅니다.

시료 간 상관 히트맵

시료 간 쌍별 피어슨 상관과 계층적 클러스터링으로, 생물학적 반복의 일관성을 평가합니다.

시료 간 PCA

차원 축소 후의 시료 분포로, 그룹 간 분리와 그룹 내 클러스터링을 보여줍니다.

차등 발현 볼케이노 플롯

log2(FoldChange)와 −log10(P)의 이중 임계값으로 상향·하향 조절 유전자를 표시하고, 주요 유전자를 라벨링합니다.

DEG 발현 패턴 클러스터링

시료 간 DEG의 발현 패턴(log10 TPM)으로, 유전자와 시료를 양방향 클러스터링합니다.

GO 기능 농축(발췌)

DEG에 대한 GO 용어(BP / CC / MF) 농축 결과와 유의성으로, 상위 용어를 표시합니다.

GO 기반 GSEA 농축 플롯

농축 점수 곡선과 순위 유전자 위치를 표시하고, NES 및 보정 p값을 주석으로 표기합니다.

KEGG 기반 GSEA 도트 플롯

KEGG 경로 농축의 유전자 비율과 유의성으로, 점 크기는 농축 유전자 수를 나타냅니다.

FAQ

How long does a Eukaryotic RNA-seq project take?

From sample QC approval, standard timelines are 3-6 weeks (library prep, sequencing and bioinformatics included); large cohorts or deep custom analyses follow the confirmed study plan.

What if my samples fail QC?

Failed QC is reported immediately with reasons, and we discuss resampling or plan adjustments with you — sequencing never proceeds without qualification.

What bioinformatics is included? Can I add custom analyses?

Standard delivery covers QC, mapping/assembly, quantification, differential and functional enrichment analyses. Custom deep-dive and multi-omics integrative analyses are supported on request.

What samples do you need for Eukaryotic RNA-seq?

Total RNA ≥1 μg with RIN ≥7 (snap-frozen, dry ice); microbial and faecal samples ≥200 mg (sterile, −80 °C); whole blood for WGS ≥500 μL (EDTA only, never heparin). RNA integrity matters more than concentration. See Technical Resources → Sample Guide.