Formalin-fixed, paraffin-embedded (FFPE) samples are common biological materials for disease diagnoses and scientific researches. Because FFPE sample tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered after decades from the original fixation, they have become an important resource for historical studies in medicine. It is, however, challenging to get intact information from such samples, as severe degradation, damage, and molecular or biological modification could occur during sample preparation and store process. 

In recent years, there were many publications about the discovery of gene variations, gene expression profiling changes, and protein biomarkers with FFPE tissues. However, there are quite few usages of next-gen sequencing (NGS) method in deciphering the FFPE nucleotide information. In order to unveil the information concealed in FFPE samples, BGI has worked relentlessly in applying NGS to FFPE samples. We expect that NGS technologies with FFPE samples will substantially facilitate our understanding of undefined pathological mechanisms and broaden our insights in biomedical research.

Benefits:

1. Abundant samples: Millions of FFPE samples stored in the world have been studied for a long time and can provide significant phenotypic information.
2. High-throughput: With 137 Illumina Hiseq 2000 instruments, BGI can provide the unprecedented throughput for FFPE research.
3. Novel variation screening: NGS can discover novel variations and detect gene expressions.
4. Comprehensive analysis: Whole genome resequencing (WGRS), whole exome sequencing (WES), transcriptome sequencing and small RNA sequencing provide various choices for different research objectives.

At BGI, we have successfully conducted many genomics and trancriptomics studies on FFPE samples, including whole genome resequencing, whole exome sequencing, RNA-Seq (Transcriptome), and small RNA sequencing. We have generated results with high concordance between FFPE and the corresponding Fresh Frozen (FF) samples.

 

Research on DNA:

1. Whole Genome Resequencing
2. Whole Exome Sequencing

Research on RNA:

1. RNA-Seq (Transcriptome)
2. Small RNA Sequencing

 

Whole Genome Resequencing

Our whole genome resequencing results suggest that the FFPE derived sequencing data has more than 90% conversion rate (clean data/raw data), indicating that the total DNA isolated with the extraction kits is of sufficient quantity and quality for sequencing using Illumina HiSeq 2000.

 

Whole Exome Sequencing

 

For whole exome sequencing, the concordance between normal SNPs (both with allele depth > 4×) and high quality SNPs ( both with allele depth > 4× and Quality Score > 20) is approximately 96%, which indicates that we can get reliable genome variations from FFPE samples comparable with results obtained from FF samples.

 

RNA-Seq (Transcriptome)

RNA extracted from FFPE samples is often of poor quality and low yield, which creates difficulties in transcriptome research. BGI has successfully used only 100 ng total RNA input of FFPE samples with different store ages and the corresponding FF samples for library construction. The mapping ratios to genome are comparable between FFPE and FF samples.

The gene expression correlation between paired FFPE/FF samples and duplicated FFPE samples shows high Pearson R (>0.89), indicating RNA-Seq (Quantification) has a good performance on FFPE samples.

 

Small RNA Sequencing

1μg of total RNA from each sample was used for library construction, and clean reads were annotated in different small RNA or mRNA degraded fragments by mapping to different non-coding RNA databases.

The data suggested that small RNA sequencing is a powerful method for miRNA and piRNA detection, and the results are similar to the ones obtained from the FF samples.

The Pearson ratio is higher than 0.99, which suggests a high technical reproducibility of miRNA detection by small RNA sequencing in FFPE samples and its high correlation to the FF controls. 

We are also testing other NGS applications on FFPE samples, including Reduced Representation Bisulfite Sequencing (RRBS) and proteome profiling, which would give researchers more flexibility in the near future. The results would enable full use of the vast number of FFPE samples available for biomedical researches and applications.

Most tumor tissue samples are preserved in the form of FFPE blocks, which in general present several challenges, including variability in fixation methods, diverse ages and sample storage environment, as well as damages that may occur to the nucleic acids during the FFPE process. To overcome the challenges of nucleic acid extraction from FFPE samples and library construction, we have developed a stable and efficient protocol and optimized the conditions at every step in our workflow.

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