Quality Control Of Libraries Research Articles

Application of the Agilent 2100 Bioanalyzer instrument as quality control for next-generation sequencing.

Next-generation sequencing (NGS) technologies have expanded the spectrum of forensic DNA analysis by facilitating efficient and precise genotyping of a large number of genetic markers. Yet, challenges persist regarding complex sample processing and assurance of equal molar concentrations across pooled samples. Since optimal cluster density is crucial for sequencing performance, the determination of both quantity and quality is indispensable for library preparation. In this study, we investigated the application of the Agilent 2100 Bioanalyzer for library quality control, as studies for forensic approaches, particularly for highly degraded postmortem samples, are rare. Our analysis encompassed assessing total DNA concentrations, fluorescence unit (FU) values, and adapter dimer concentrations in purified DNA libraries derived from buccal swabs and tissue samples of decomposed corpses. The sensitivity study tested a serial dilution derived from buccal swabs and revealed a decrease in FU values and an increase in adapter dimers with declining DNA input concentrations. Deviations in total DNA concentrations and average peak heights between the Agilent 2100 Bioanalyzer runs indicated a lack of repeatability in data and presented challenges in accurate quantification, which was also observed in previous studies. Yet, the analysis of degraded samples from decomposed human remains has shown the ability to detect adapter dimer concentrations, which can be crucial for the quality of subsequent NGS library preparation and sequencing success. Therefore, the Agilent 2100 Bioanalyzer proves to be a valuable tool for NGS quality control.

Longitudinal Analysis of Circulating Tumor DNA (ctDNA) in Diffuse Large B-Cell Lymphoma (DLBCL) Using KAPA HyperCap Design Share Non-Hodgkin Lymphoma (KAPA HyperCap DS NHL) Next-Generation Sequencing (NGS) Panel

Introduction Studies have shown that dynamic changes in lymphoma ctDNA levels are associated with patient outcomes and that monitoring lymphoma ctDNA may help identify patients who are at risk of refractory disease or relapse. NGS panels can successfully identify patient-specific tumor reporter variants in plasma specimens using a tissue-naïve approach that avoids limitations of patient tissue availability. Recently, using the AVENIO Oncology Assay (AOA) NHL Test* validated by the Roche Molecular CAP/CLIA Laboratory, a pre-specified analysis plan using samples from ~800 DLBCL patients from the POLARIX study validated ctDNA as an early prognostic biomarker (Herrera et al., Blood 2022). To further support clinical research on early molecular response (EMR) to treatment and minimal residual disease (MRD) in NHL, we introduce a KAPA HyperCap DS NHL panel designed to cover 100% of the target regions in the AOA NHL Test used for the POLARIX study. We describe the NGS workflow and bioinformatics analysis suitable for identification and monitoring of ctDNA using a tissue-naïve approach. We further describe performance characterization studies and feasibility of ctDNA detection using plasma specimens from patients with DLBCL. Methods KAPA HyperCap DS NHL panel covers coding and/or untranslated regions of 383 genes, plus additional intergenic regions, for a total of 341 Kb. This panel is used in combination with KAPA HyperCap workflows and KAPA reagents on Illumina® platforms, to sequence plasma cfDNA and matched genomic (g)DNA to identify tumor-specific single-nucleotide variants (SNVs) and monitor the dynamics of ctDNA. ctDNA detection and monitoring are supported by open-source bioinformatics tools for a fully integrated MRD analysis solution. Contrived samples for workflow characterization were comprised of commercially available reference materials with known SNV allele frequencies (AF) mimicking cfDNA (SeraSeq® Complete Mutation Mix and Twist Pan-cancer Reference Standard), as well as pre-characterized healthy donor cfDNA. Clinically annotated DLBCL samples were characterized and serially diluted to demonstrate feasibility of longitudinal mutation analysis. Results Serially diluted cfDNA samples and high molecular weight gDNA samples were used to assess the performance of the plasma cfDNA and germline workflows. Library quality control metrics met the yield and size distribution criteria for sequencing. A median of 88 M raw reads were obtained across all libraries. De-duplication yielded a median coverage depth range from 5000-9100 across samples. Median on-target rate (% selected bases) was between 74% and 80%. Average error rate was between 0.00024 or 0.00031 mismatches/read depth. 9 and 3 variants were detected after germline and blocklist filtering in the two commercial reference samples. Variant calling sensitivity was 100% across replicates at 5, 1 and 0.5% AF. In the serial dilution analysis, ctDNA was detected in all replicates at 5, 0.1 and 0.05% mean AF. For samples at 0.01% mean AF, ctDNA detection sensitivity was 83%. Initial data demonstrate that 131 SNV reporters were detected at 23% mean AF in a plasma sample from a treatment-naïve DLBCL patient. 4 SNV reporters were detected at 1.9% mean AF in a plasma sample from an immunochemotherapy-treated DLBCL patient. After 10x and 500x dilution, ctDNA positivity was accurately called at 2.3% and 0.05% mean AF levels for the patient with 131 reporters, and at 0.19% mean AF level for the patient with 4 reporters (Monte Carlo p-value <0.0001). Conclusion The use of KAPA HyperCap DS NHL panel with KAPA HyperCap workflows and open-source bioinformatics tools enables the detection and monitoring of ctDNA for NHL research applications. In contrived samples, variants were detected with high reproducibility at AF as low as 0.05% and with good reproducibility at AF of 0.01%. For a DLBCL patient with 131 SNV reporters in plasma, ctDNA was detectable down to 0.05% mean AF levels, while for a DLBCL patient with 4 SNV reporters, ctDNA was detectable down to 0.19% mean AF levels, demonstrating that sensitivity of Monte Carlo-based ctDNA detection depends on the number of reporter variants. *AVENIO Oncology Assay (AOA) NHL Test and KAPA HyperCap Design Share panels are for Research Use Only, not for use in diagnostic procedures. AVENIO and KAPA are trademarks of Roche. All other product names and trademarks are the property of their respective owners.

PSVIII-22 Maternal vitamin and mineral supplementation affect fetal hepatic expression of genes underlying mineral homeostasis and lipid metabolism in early pregnancy

Abstract Vitamins and minerals play critical roles in functions such as hormone production, DNA synthesis, regulation of gene expression, and lipid metabolism. However, the impact of vitamin and mineral supplementation on fetal programming and the interplay with gene expression of fetal organs remains unclear. We used a differential gene expression analysis to determine effects of maternal vitamin and mineral supplementation (from pre-breeding to d 83 post-breeding) on fetal hepatic gene expression and the pathways underlying liver function and metabolism at 83 d of gestation. Crossbred Angus beef heifers were supplemented (VTM, n = 7) or not (CON, n = 8) with 113 g•heifer-1•d-1 of mineral premix (Purina® Wind & Rain Storm All-Season 7.5 Complete) from a minimum d 71 before breeding through d 83 of gestation. After breeding, heifers were fed to gain 0.79 kg/d. All heifers were surgically ovariohysterectomized on d 83 and fetal liver collected. Total RNA was isolated from the fetal liver (n = 15) and gene expression measured with RNA-Seq. After library quality control and read mapping, differential expression was performed using edgeR. We identified 53 genes upregulated and 37 downregulated in the VTM group (adj.Pval < 0.1). Genes involved with mineral homeostasis, such as MT1A, MT1E, and MT2A, were among those differentially expressed underlying the mineral absorption pathway. ABCA1 and ABCA6, which are involved in cholesterol and metal ion transport across the plasma membrane, and PPARG and SDR16C5, that act on lipoprotein transport and metabolism, were upregulated in the VTM group. Also upregulated in the VTM group, the CUBN gene plays a role in vitamin and iron metabolism. In summary, maternal vitamin and mineral supplementation from pre-breeding to d 83 of gestation leads to upregulation of fetal hepatic genes acting on mineral homeostasis, lipid transport, and metabolism.

Fast and Efficient 5'P Degradome Library Preparation for Analysis of Co-Translational Decay in Arabidopsis.

The recent development of high-throughput technologies based on RNA sequencing has allowed a better description of the role of post-transcriptional regulation in gene expression. In particular, the development of degradome approaches based on the capture of 5′monophosphate decay intermediates allows the discovery of a new decay pathway called co-translational mRNA decay. Thanks to these approaches, ribosome dynamics could now be revealed by analysis of 5′P reads accumulation. However, library preparation could be difficult to set-up for non-specialists. Here, we present a fast and efficient 5′P degradome library preparation for Arabidopsis samples. Our protocol was designed without commercial kit and gel purification and can be easily done in one working day. We demonstrated the robustness and the reproducibility of our protocol. Finally, we present the bioinformatic reads-outs necessary to assess library quality control.

Performing Ribosome Profiling to Assess Translation in Vegetative and Meiotic Yeast Cells.

Ribosome profiling, first developed in 2009, is the gold standard for quantifying and qualifying changes to translation genome-wide (Ingolia et al., Science, 2009). Though first designed and optimized in vegetative budding yeast, it has since been modified and specialized for use in diverse cellular states in yeast, as well as in bacteria, plants, human cells, and many other organisms (Ingolia et al. Science, 2009, reviewed in (Ingolia et al., Cold Spring Harb Perspect Biol, 2019; Brar and Weissman, Nat Rev Mol Cell Biol, 2015)). Here we report the current ribosome profiling protocol used in our lab to study genome-wide changes to translation in budding yeast undergoing the developmental process of meiosis (Brar et al., Science, 2012; Cheng et al., Cell, 2018). We describe this protocol in detail, including the following steps: collection and flash freezing samples, cell lysis and extract preparation, sucrose gradient centrifugation and monosome collection, RNA extraction, library preparation, and library quality control. Almost every step presented here should be directly applicable to performing ribosome profiling in other eukaryotic cell types or cell states.

PSIII-42 Genome-wide expression profile of trophoblastic cells during late pregnancy in ewes

Abstract During pregnancy, the placenta plays a pivotal role in fetal-maternal communication. Despite the known involvement of trophoblast mononuclear (MNC) and binuclear (BNC) cells in placentation and fetal development, the regulatory mechanisms underlying the gene expression in these cells and their role in pregnancy have not been fully elucidated. Thus, a genome-wide expression profile analysis of MNC and BNC from ovine placentomes on day 90 and 130 of pregnancy was performed to identify differentially expressed genes (DEG) and regulatory pathways. After tissue separation, enzymatic digestion, and elutriation, MNC and BNC were obtained, and the total RNA was isolated and sequenced (n = 2 ewes/day). Differential expression analysis was carried out with DESeq2 after library quality control and read mapping using FastQC and STAR, respectively. Among six pairwise contrasts possible, herein, we focused on the one between MNC and BNC on day 130. Accordingly, we identified 514 genes upregulated and 161 downregulated in BNC (adj.Pval < 0.05). Interestingly, we found nine DE long intergenic noncoding RNAs (lincRNA). Although the function of lincRNAs remains under investigation, its transcription has been associated with gene expression regulation in utero. The DEGs functional over-representation analysis included Rap1 and PI3k-Akt signaling pathways as up-regulated in BNC (adj.Pval. < 0.05). These pathways are activated by extracellular signals and are involved in cell proliferation and differentiation, morphogenesis, and energy metabolism. Furthermore, we identified PLA2G10 gene that codes a phospholipase protein involved in the production of prostaglandins, which have been associated with placental blood flow. These findings suggest an intricate and complex network underlying gene expression between cells and time points that are essential for the fetal-maternal placenta relationship. This project was supported by Agriculture and Food Research Initiative Competitive Grant n. 2016-67016-24884 from the USDA National Institute of Food and Agriculture.

DIALib-QC an assessment tool for spectral libraries in data-independent acquisition proteomics

Data-independent acquisition (DIA) mass spectrometry, also known as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH), is a popular label-free proteomics strategy to comprehensively quantify peptides/proteins utilizing mass spectral libraries to decipher inherently multiplexed spectra collected linearly across a mass range. Although there are many spectral libraries produced worldwide, the quality control of these libraries is lacking. We present the DIALib-QC (DIA library quality control) software tool for the systematic evaluation of a library’s characteristics, completeness and correctness across 62 parameters of compliance, and further provide the option to improve its quality. We demonstrate its utility in assessing and repairing spectral libraries for correctness, accuracy and sensitivity.

SOLQC: Synthetic Oligo Library Quality Control tool.

Recent years have seen a growing number and an expanding scope of studies using synthetic oligo libraries for a range of applications in synthetic biology. As experiments are growing by numbers and complexity, analysis tools can facilitate quality control and support better assessment and inference. We present a novel analysis tool, called SOLQC, which enables fast and comprehensive analysis of synthetic oligo libraries, based on NGS analysis performed by the user. SOLQC provides statistical information such as the distribution of variant representation, different error rates and their dependence on sequence or library properties. SOLQC produces graphical reports from the analysis, in a flexible format. We demonstrate SOLQC by analyzing literature libraries. We also discuss the potential benefits and relevance of the different components of the analysis. SOLQC is a free software for non-commercial use, available at https://app.gitbook.com/@yoav-orlev/s/solqc/. For commercial use please contact the authors. Supplementary data are available at Bioinformatics online.

High-Throughput Cellular RNA Sequencing (HiCAR-Seq): Cost-Effective, High-Throughput 3' mRNA-Seq Method Enabling Individual Sample Quality Control.

High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.

Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq.

Single-cell RNA-sequencing (scRNA-seq) enables a comprehensive analysis of the transcriptome of individual cells by next-generation sequencing. ScRNA-seq offers an unbiased approach to investigate the cellular heterogeneity and dynamics of diverse biological systems, including the immune system. Optimization of the technical procedures performed prior to RNA-seq analysis is imperative to the success of a scRNA-seq experiment. Here, three major experimental procedures are described: (1) the isolation of immune CD8a+ T cells from primary murine tissue, (2) the generation of single-cell cDNA libraries using the 10× Genomics Chromium Controller and the Chromium Single Cell 3' Solution, and (3) cDNA library quality control. In this protocol, CD8a+ T cells are isolated from murine spleen tissue, but any cell type of interest can be enriched and used for single-cell cDNA library generation and subsequent RNA-seq experiments.

Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications.

Next-generation DNA-sequencing (NGS) technologies, which are designed to streamline the acquisition of massive amounts of sequencing data, are nonetheless dependent on various preparative steps to generate DNA fragments of required concentration, purity and average size (molecular weight). Current automated electrophoresis systems for DNA- and RNA-sample quality control, such as Agilent’s Bioanalyzer® and TapeStation® products, are costly to acquire and use; they also provide limited information for samples having broad size distributions. Here, we describe a software tool that helps determine the size distribution of DNA fragments in an NGS library, or other DNA sample, based on gel-electrophoretic line profiles. The software, developed as an ImageJ plug-in, allows for straightforward processing of gel images, including lane selection and fitting of univariate functions to intensity distributions. The user selects the option of fitting either discrete profiles in cases where discrete gel bands are visible or continuous profiles, having multiple bands buried under a single broad peak. The method requires only modest imaging capabilities and is a cost-effective, rigorous alternative characterization method to augment existing techniques for library quality control.

The impact of common PCR inhibitors on forensic MPS analysis

Massively parallel sequencing holds great promise for new possibilities in the field of forensic genetics, enabling simultaneous analysis of multiple markers as well as offering enhanced short tandem repeat allele resolution. A challenge in forensic DNA analysis is that the samples often contain low amounts of DNA in a background that may interfere with downstream analysis. PCR inhibition mechanisms of some relevant molecules have been studied applying e.g. real-time PCR and digital PCR. However, a detailed understanding of the effects of inhibitory molecules on forensic MPS, including mechanisms and ways to relieve inhibition, is missing. In this study, the effects of two well-characterized PCR inhibitors, humic acid and hematin, have been studied using the ForenSeq DNA Signature Prep kit. Humic acid and hematin resulted in lowered read numbers as well as specific negative effects on certain markers. Quality control of libraries with Fragment analyzer showed that increasing amounts of inhibitors caused a lowered amplicon quantity and that the larger amplicons were more likely to drop out. Further, the inhibitor tolerance could be improved 5–10 times by addition of bovine serum albumin in the initial PCR. On the contrary to the samples with inhibitors, low-template samples resulted in lowered read numbers for all markers. This difference strengthened the conclusion that the inhibitors have a negative effect on the DNA polymerase activity in the initial PCR. Additionally, a common capillary gel electrophoresis-based STR kit was shown to handle at least 200 times more inhibitors than the ForenSeq DNA Signature Prep kit. This suggests that there is room for improvement of the PCR components to ensure analytical success for challenging samples, which is needed for a broad application of MPS for forensic STR analysis.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Agilent buys capillary electrophoresis expert

Agilent Technologies has signed an agreement to acquire Advanced Analytical Technologies, a capillary electrophoresis expert, for $250 million. Buying the Iowa State University spin-off, Agilent says, will give it a leg up in the market for a widely used separation technique to analyze nucleic acids. Advanced Analytical also brings an alliance with next-generation gene sequencing leader Illumina, providing Agilent with “the broadest market share position in the NGS library quality control space,” points out Leerink Research analyst Puneet Souda.

Hichipper: a preprocessing pipeline for calling DNA loops from HiChIP data.

Mumbach et al. recently described HiChIP, a novel protein-mediated chromatin conformation assay that lowers cellular input requirements while simultaneously increasing the yield of informative reads compared to previous methods. To facilitate the dissemination and adoption of this assay, we introduce hichipper (http://aryeelab.org/hichipper), an open-source HiChIP data preprocessing tool, with features that include bias-corrected peak calling, library quality control, DNA loop calling, and output of processed data for downstream analysis and visualization.

Genome Sequencing of Steroid-Producing Bacteria with Illumina Technology.

Illumina technology is widely used for bacterial whole-genome sequencing due to its simplicity, cheapness, reliability, and abundant software for manipulation with raw data. Illumina technology belongs to a second generation of whole genome sequencing that yields great amount of short reads for genome regions. Genomic DNA is fragmented to short pieces. DNA fragments are amplified for signal increasing, and are read using sequencing-by-synthesis. Millions of short reads up to 100-300 bp in length are assembled in continuous sequences. Mate-pair technology allows resolving a long repeat.Here, we describe the principles of standard and mate-pair library preparation from DNA samples, library quality control, sequencing with MiSeq instrument and following data bioinformatics treatment. Software for genome assembly and completion are listed that assemble, map, annotate, visualize, edit and allow doing other manipulations with genomic sequences. The whole genomes sequencing of the steroid-producing Actinobacteria using these protocols is exemplified.

Genome-wide functional analysis using the barcode sequence alignment and statistical analysis (Barcas) tool.

BackgroundPooled library screen analysis using shRNAs or CRISPR-Cas9 hold great promise to genome-wide functional studies. While pooled library screens are effective tools, erroneous barcodes can potentially be generated during the production of many barcodes. However, no current tools can distinguish erroneous barcodes from PCR or sequencing errors in a data preprocessing step.ResultsWe developed the Barcas program, a specialized program for the mapping and analysis of multiplexed barcode sequencing (barcode-seq) data. For fast and efficient mapping, Barcas uses a trie data structure based imperfect matching algorithm which generates precise mapping results containing mismatches, shifts, insertions and deletions (indel) in a flexible manner. Barcas provides three functions for quality control (QC) of a barcode library and distinguishes erroneous barcodes from PCR or sequencing errors. It also provides useful functions for data analysis and visualization.ConclusionsBarcas is an all-in-one package providing useful functions including mapping, data QC, library QC, statistical analysis and visualization in genome-wide pooled screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1326-9) contains supplementary material, which is available to authorized users.

Simultaneous Digital Quantification and Fluorescence-Based Size Characterization of Massively Parallel Sequencing Libraries

Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.

The use of EST expression matrixes for the quality control of gene expression data.

EST expression profiling provides an attractive tool for studying differential gene expression, but cDNA libraries' origins and EST data quality are not always known or reported. Libraries may originate from pooled or mixed tissues; EST clustering, EST counts, library annotations and analysis algorithms may contain errors. Traditional data analysis methods, including research into tissue-specific gene expression, assume EST counts to be correct and libraries to be correctly annotated, which is not always the case. Therefore, a method capable of assessing the quality of expression data based on that data alone would be invaluable for assessing the quality of EST data and determining their suitability for mRNA expression analysis. Here we report an approach to the selection of a small generic subset of 244 UniGene clusters suitable for identification of the tissue of origin for EST libraries and quality control of the expression data using EST expression information alone. We created a small expression matrix of UniGene IDs using two rounds of selection followed by two rounds of optimisation. Our selection procedures differ from traditional approaches to finding “tissue-specific” genes and our matrix yields consistency high positive correlation values for libraries with confirmed tissues of origin and can be applied for tissue typing and quality control of libraries as small as just a few hundred total ESTs. Furthermore, we can pick up tissue correlations between related tissues e.g. brain and peripheral nervous tissue, heart and muscle tissues and identify tissue origins for a few libraries of uncharacterised tissue identity. It was possible to confirm tissue identity for some libraries which have been derived from cancer tissues or have been normalised. Tissue matching is affected strongly by cancer progression or library normalisation and our approach may potentially be applied for elucidating the stage of normalisation in normalised libraries or for cancer staging.

Building and Searching Tandem Mass Spectral Libraries for Peptide Identification

Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. Conceptually, the premise of this approach is that the tandem MS fragmentation pattern of a peptide under some fixed conditions is a reproducible fingerprint of that peptide, such that unknown spectra acquired under the same conditions can be identified by spectral matching. In actual practice, a spectral library is first meticulously compiled from a large collection of previously observed and identified tandem MS spectra, usually obtained from shotgun proteomics experiments of complex mixtures. Then, a query spectrum is then identified by spectral matching using recently developed spectral search engines. This review discusses the basic principles of the two pillars of this approach: spectral library construction, and spectral library searching. An overview of the software tools available for these two tasks, as well as a high-level description of the underlying algorithms, will be given. Finally, several new methods that utilize spectral libraries for peptide identification in ways other than straightforward spectral matching will also be described.