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actions-user committed Sep 9, 2024
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84 changes: 35 additions & 49 deletions 1- Library preparation.html
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Expand Up @@ -201,88 +201,77 @@ <h2>RNA library bias<a class="headerlink" href="#rna-library-bias" title="Link t
<a class="reference internal image-reference" href="_images/protocol_RNA-seq_library_bias_vanDjik_etal_2014.png"><img alt="*source: https://doi.org/10.1016/j.yexcr.2014.01.008*" class="align-center" src="_images/protocol_RNA-seq_library_bias_vanDjik_etal_2014.png" style="width: 400px;" /></a>
<section id="sample-preservation-and-isolation">
<h3><strong>Sample Preservation and Isolation</strong><a class="headerlink" href="#sample-preservation-and-isolation" title="Link to this heading"></a></h3>
<ol class="arabic">
<li><p>Degradation of RNA:</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple">
<li><p>Degradation of RNA:</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>Minimizing the sample processing and freezing and thawing cycles, ensures that RNA is preserved as best as possible.</p>
</div>
</div></blockquote>
</li>
<li><p>RNA extraction:</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="2">
<li><p>RNA extraction:</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>If possible use high concentrations of RNA samples or avoid TRIzol extraction altogether.</p>
</div>
</div></blockquote>
</li>
</ol>
</section>
<section id="library-construction">
<h3><strong>Library Construction</strong><a class="headerlink" href="#library-construction" title="Link to this heading"></a></h3>
<ol class="arabic">
<li><p><strong>Low-quality and/or low-quantity RNA samples</strong>:</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple">
<li><p><strong>Low-quality and/or low-quantity RNA samples</strong>:</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>RNase H has been the best method for detecting low-qualityRNA and even could effectively replace the standard RNA-seq method based on oligo (dT).
For low-quantity RNA,the SMART and NuGEN approaches had lower duplication rates and significantly decreased the necessary amount of starting material compared to other methods.</p>
</div>
</div></blockquote>
</li>
<li><p><strong>mRNA enrichment bias</strong>: In eukaryotes enrich for polyadenylated RNA transcripts with oligo (dT) primers have shown that this method remove all non-poly (A) RNAs, such a reolication-dependant histones and lncRNAs (lacking of polyA),or incomplete mRNAs.</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="2">
<li><p><strong>mRNA enrichment bias</strong>: In eukaryotes enrich for polyadenylated RNA transcripts with oligo (dT) primers have shown that this method remove all non-poly (A) RNAs, such a reolication-dependant histones and lncRNAs (lacking of polyA),or incomplete mRNAs.</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>Targeting rRNA as depletion method will not limit to only mRNA molecules, may capture more immature transcripts, leading to a complexity increase of sequencing data (also is more expensive).
Subtractive hybridization using rRNA-specific probes as the method that introduced the least bias in relative transcript abundance,</p>
</div>
</div></blockquote>
</li>
<li><p><strong>RNA fragmentation bias</strong>: There are two major approaches of RNA fragmentation: chemical (using metal ions) and enzymatic (using RNase III). During this process could be introduced lenght biases or errors (propagated to later cycles).</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="3">
<li><p><strong>RNA fragmentation bias</strong>: There are two major approaches of RNA fragmentation: chemical (using metal ions) and enzymatic (using RNase III). During this process could be introduced lenght biases or errors (propagated to later cycles).</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>Studies have shown that methods that involve non specific restriction endonucleases indicate less sequence bias and have been shown to perform similarly to the physical methods. Also enzymatic methoda are easy to automate</p>
</div>
</div></blockquote>
</li>
<li><p><strong>Primer bias</strong>: During reverse transcription into cDNA by random hexamers can lead to deviation of nucleotide content of RNA sequencing reads, resulting in low complexity of RNA sequencing data.</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="4">
<li><p><strong>Primer bias</strong>: During reverse transcription into cDNA by random hexamers can lead to deviation of nucleotide content of RNA sequencing reads, resulting in low complexity of RNA sequencing data.</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>Could be avoid using the Illumina Genome Analyzer, which perform the reverse transcription directly on the flowcells, avoiding the PCR.
Also has been proposed a bioinformatics tool in a reweighing scheme to adjust for the bias and make the distribution of the reads more uniform.</p>
</div>
</div></blockquote>
</li>
<li><p><strong>Adapter ligation bias</strong>: Adapter ligation introduces a significant but widely overlooked bias in the results of NGS small RNA sequencing.</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="5">
<li><p><strong>Adapter ligation bias</strong>: Adapter ligation introduces a significant but widely overlooked bias in the results of NGS small RNA sequencing.</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>As a solution, several groups propose to randomize the 3’ end of the 5’adapter and the 5’end of the 3’adapter.
The strategy is based on the hypothesis that a population of degenerate adapters would average out the sequencing bias because the slightly different adapter molecules would form stable secondary structures with a more diverse population of RNAsequences - Reverse transcription bias: reverse transcriptases tend to produce false second strand cDNA throughDNA-dependent DNA polymerase. ActinomycinD, a compound that specifically inhibits DNA-dependent DNAsynthesis, has been proposed as an agent to eliminate antisense artifacts</p>
</div>
</div></blockquote>
</li>
<li><p><strong>Reverse Transcription</strong>: A known feature of reverse transcriptases is that they tend to produce false second strand cDNA through DNA-dependent DNA polymerase. This may not be able to distinguish the sense and antisense transcript and create difficulties for the data analysis.</p>
<blockquote>
<div><div class="admonition tip">
<ol class="arabic simple" start="6">
<li><p><strong>Reverse Transcription</strong>: A known feature of reverse transcriptases is that they tend to produce false second strand cDNA through DNA-dependent DNA polymerase. This may not be able to distinguish the sense and antisense transcript and create difficulties for the data analysis.</p></li>
</ol>
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<ul class="simple">
<li><p>The deoxyuridine triphosphate (dUTP) method, one of the leading cDNA-based strategies, can be specifically removed by enzymatic digestion</p></li>
<li><p>Another method is to synthesize the first strand of cDNA using labeled random hexamer primer and SSS using DNA-RNA template-switching primer</p></li>
</ul>
</div>
</div></blockquote>
</li>
<ol class="arabic" start="7">
<li><p><strong>PCR amplification bias</strong>: main source of artifacts and base composition bias in the process of library construction:</p>
<blockquote>
<div><p>7.1. Extremely AT/GC-Rich: Fragments of GC-neutral can be amplified more than GC-rich or AT-rich fragments.</p>
<blockquote>
<div><div class="admonition tip">
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<ul class="simple">
<li><p>Through the use of custom adapters, the samples without amplification and ligation can be hybridized directly with the oligonucleotides on the flowcell surface, thus avoiding the biases and duplicates of PCR.</p></li>
Expand All @@ -293,15 +282,12 @@ <h3><strong>Library Construction</strong><a class="headerlink" href="#library-co
a number of additives have been reported to play an important role in reducing the bias of PCR ampli-fication, including small amides such as formamide, small sulfoxides such as dimethyl sulfoxide (DMSO),
or reducingcompounds such as β-mercaptoethanol or dithiothreitol(DTT).</p>
</div>
</div></blockquote>
<p>7.2. PCR cyle: PCR can exponentially amplify DNA/cDNA templates, thus leading to a significant increase of amplification bias with the number of PCR cycles.</p>
<blockquote>
<div><div class="admonition tip">
<div class="admonition tip">
<p class="admonition-title">Tip</p>
<p>it is recommended that PCR be performedusing as few cycle numbers as possible to mitigation bias.</p>
</div>
</div></blockquote>
</div></blockquote>
</li>
</ol>
<div class="admonition seealso">
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11 changes: 1 addition & 10 deletions 2- Sequencing technologies.html
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Expand Up @@ -161,27 +161,18 @@ <h2>FASTQ format and Phred quality score<a class="headerlink" href="#fastq-forma
<a class="reference internal image-reference" href="_images/fastq_format.png"><img alt="_images/fastq_format.png" class="align-center" src="_images/fastq_format.png" style="width: 400px;" /></a>
<p>For each read, the information it’s divided in four lines:</p>
<blockquote>
<div><blockquote>
<div><blockquote>
<div><ol class="arabic simple">
<li><p>Sequence identifier: starts with ‘&#64;’ and contains information about the read. Such as the instrument, run ID, flow cell ID, lane, tile, x, y coordinates, and read number.</p></li>
</ol>
</div></blockquote>
<div class="admonition note">
<p class="admonition-title">Note</p>
<p>The &#64; symbol can not be used for count the number of reads, because it could also appear as a quality score symbol.</p>
</div>
</div></blockquote>
<ol class="arabic simple" start="2">
<li><dl class="simple">
<dt>Sequence: the nucleotide sequence of the read.</dt><dd><ol class="arabic simple" start="3">
<li><p>Sequence: the nucleotide sequence of the read.</p></li>
<li><p>Quality identifier: starts with ‘+’ and contains the same information as the sequence identifier. Or it may be empty and in some cases is used for metadata.</p></li>
<li><p>Quality scores: the Phred quality score for each base in the read. The Phred quality score is a measure of the quality of the base call,</p></li>
</ol>
</dd>
</dl>
</li>
</ol>
<blockquote>
<div><div class="math notranslate nohighlight">
\[Q = -10 * log10(P)\]</div>
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18 changes: 10 additions & 8 deletions 3- Quality Control and Preprocessing.html
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Expand Up @@ -144,13 +144,15 @@ <h4>FASTQC<a class="headerlink" href="#fastqc" title="Link to this heading"><
<li><p><strong>Per sequence GC content</strong>: GC content distribution for all the reads in the file, and compared to a modelled normal distribution of human GC content (blue line).</p>
<blockquote>
<div><a class="reference internal image-reference" href="_images/Per_seq_GC_content.png"><img alt="*Per Sequence GC Content FASTQC module*" class="align-center" src="_images/Per_seq_GC_content.png" style="width: 400px;" /></a>
</div></blockquote>
</li>
</ol>
<div class="admonition danger">
<p class="admonition-title">Danger</p>
<p>If the GC content is not close to the normal distribution, or more than one peak is found, this could indicate a contamination or a problem in the library preparation.
Also, depending on the organism the GC content could vary, so if possible it’s good to know the GC content of the organism of interest previously and avoid compare it with the human modelled distribution.</p>
</div>
</div></blockquote>
</li>
<ol class="arabic" start="7">
<li><p><strong>Per Base N content</strong>: If the sequencer is unable to determine the base in a position, it will be represented as an ‘N’. This section shows the distribution of Ns in the reads.</p>
<blockquote>
<div><a class="reference internal image-reference" href="_images/Per_base_N_content.png"><img alt="*Per Base N Content FASTQC module*" class="align-center" src="_images/Per_base_N_content.png" style="width: 400px;" /></a>
Expand Down Expand Up @@ -195,8 +197,8 @@ <h4>FASTQ-Screen<a class="headerlink" href="#fastq-screen" title="Link to this h
<div><ul class="simple">
<li><p>PhiX: is a control used by Illumina to check the quality of the sequencing run (if the library is under or overloaded).</p></li>
<li><p>rRNA: in RNA-seq is a good control of rRNA depletion during library preparation.</p></li>
<li><p>Lambda</p></li>
<li><p>Vectors: to check that vectors used during library preprartion have not been amplified.</p></li>
<li><p>Lambda: cloning vector.</p></li>
<li><p>Vectors: other vectors used during library preprartion.</p></li>
<li><p>Adapters</p></li>
</ul>
</div></blockquote>
Expand All @@ -223,10 +225,10 @@ <h3>Pre-processing<a class="headerlink" href="#pre-processing" title="Link to th
<p>Typical tools used for pre-processing are:</p>
<blockquote>
<div><ul class="simple">
<li><p>Trimmomatic &lt;<a class="reference external" href="http://www.usadellab.org/cms/index.php?page=trimmomatic">http://www.usadellab.org/cms/index.php?page=trimmomatic</a>&gt;</p></li>
<li><p>Cutadapt, only remove the adapaters (it needs to be used in combination with sickle), requires the adapter sequence to be known &lt;<a class="reference external" href="https://cutadapt.readthedocs.io/en/stable/">https://cutadapt.readthedocs.io/en/stable/</a>&gt;</p></li>
<li><p>Sickle, remove low quality tail bases &lt;<a class="reference external" href="https://github.com/najoshi/sickle">https://github.com/najoshi/sickle</a>&gt;</p></li>
<li><p>fastp &lt;<a class="reference external" href="https://github.com/OpenGene/fastp">https://github.com/OpenGene/fastp</a>&gt;</p></li>
<li><p>Trimmomatic <a class="reference external" href="http://www.usadellab.org/cms/index.php?page=trimmomatic">http://www.usadellab.org/cms/index.php?page=trimmomatic</a>.</p></li>
<li><p>Cutadapt, only remove the adapaters (it needs to be used in combination with sickle), requires the adapter sequence to be known <a class="reference external" href="https://cutadapt.readthedocs.io/en/stable/">https://cutadapt.readthedocs.io/en/stable/</a>.</p></li>
<li><p>Sickle, remove low quality tail bases <a class="reference external" href="https://github.com/najoshi/sickle">https://github.com/najoshi/sickle</a>.</p></li>
<li><p>FASTP <a class="reference external" href="https://github.com/OpenGene/fastp">https://github.com/OpenGene/fastp</a>.</p></li>
</ul>
</div></blockquote>
<p>Fastp performs in all one the following corrections:</p>
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