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<li class="toctree-l1"><a class="reference internal" href="about.html">About the course</a></li>
<li class="toctree-l1 current"><a class="current reference internal" href="#">1 Library preparation</a><ul>
<li class="toctree-l2"><a class="reference internal" href="#main-causes-of-poor-quality-data">Main Causes of poor quality data</a></li>
<li class="toctree-l2"><a class="reference internal" href="#id1">Library preparation</a></li>
<li class="toctree-l2"><a class="reference internal" href="#library-preparation-bias">Library preparation bias</a><ul>
<li class="toctree-l3"><a class="reference internal" href="#dna-library-bias">DNA library bias</a></li>
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<li class="toctree-l2"><a class="reference internal" href="#short-reads-sequencing-illumina">Short Reads sequencing (Illumina)</a><ul>
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</div></blockquote>
<p>The html report generated for each file its divided in the following modules:</p>
<blockquote>
<div><p>#1. <strong>Basic Statistics</strong>: display the information related with the file, number and leght of the sequences, and overall %GC.
#2. <strong>Per base sequence quality</strong>: shows how the quality score (y axis) varys throughout the sequence reads (x axis). For each position a BoxWhisker is displayed, the red line represents the median and the blue the mean. Commonly the quality score tend to decrease at the end of the reads, because the polymerase tends to make more mistakes as the read progresses.</p>
<blockquote>
<div><p>is the median os any base is less than 25 a warning will arise.</p>
</div></blockquote>
<p>#3. <strong>Per tile sequence quality</strong>: shows the quality score distribution for each tile in the flowcell.
#4. <strong>Per sequence quality score</strong>: shows the distribution of the quality scores for all the reads in the file. If a huge amount of reads subset have a poor average quality this could indicate a systematic problem.
#5. <strong>Per base sequence content</strong>: proportion of each base position for the four nucleotides. A strong bias in the nucleotide composition could indicate a problem in the library preparation.
#6. <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.</p>
</div></blockquote>
<div><ol class="arabic simple">
<li><p><strong>Basic Statistics</strong>: display the information related with the file, number and leght of the sequences, and overall %GC.</p></li>
<li><dl class="simple">
<dt><strong>Per base sequence quality</strong>: shows how the quality score (y axis) varys throughout the sequence reads (x axis). For each position a BoxWhisker is displayed, the red line represents the median and the blue the mean. Commonly the quality score tend to decrease at the end of the reads, because the polymerase tends to make more mistakes as the read progresses.</dt><dd><p>is the median os any base is less than 25 a warning will arise.</p>
</dd>
</dl>
</li>
<li><p><strong>Per tile sequence quality</strong>: shows the quality score distribution for each tile in the flowcell.</p></li>
<li><p><strong>Per sequence quality score</strong>: shows the distribution of the quality scores for all the reads in the file. If a huge amount of reads subset have a poor average quality this could indicate a systematic problem.</p></li>
<li><p><strong>Per base sequence content</strong>: proportion of each base position for the four nucleotides. A strong bias in the nucleotide composition could indicate a problem in the library preparation.</p></li>
<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.</p></li>
</ol>
<div class="admonition danger">
<p class="admonition-title">Danger</p>
<blockquote>
<div><p>If the GC content is not close to the normal distribution, this could indicate a contamination or a problem in the library preparation.
<p>If the GC content is not close to the normal distribution, this could indicate a contamination or a problem in the library preparation.
Also, depending on the organism the GC content could vary, so it is important to know the GC content of the organism of interest (so avoid comparison with reference curve).</p>
</div></blockquote>
<p>#7. <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.
#8. <strong>Sequence Lenght Distribution</strong>: distribution of fragment sizes, for delimited size lenght (number of cycles) a peak only at one size is observed.
#9. <strong>Duplicate Sequences</strong>: shows the number of duplicated sequences in the file. a high level of duplication could indicate a enrichment bias (i.e. PCR amplification). Low level of duplication may indicate a very high level of coverage of the target sequence.
#10. <strong>Overrepresented sequences</strong>: show in a single sequence is very overrepresented in the file. This could indicate a contamination or a problem in the library preparation.
#11. <a href="#id1"><span class="problematic" id="id2">**</span></a>Adapter content <a href="#id3"><span class="problematic" id="id4">**</span></a>: shows the presence of adapter sequences in the reads. If there is presence of adapters, the reads should be trimmed before further analysis.</p>
</div>
<ol class="arabic simple" 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></li>
<li><p><strong>Sequence Lenght Distribution</strong>: distribution of fragment sizes, for delimited size lenght (number of cycles) a peak only at one size is observed.</p></li>
<li><p><strong>Duplicate Sequences</strong>: shows the number of duplicated sequences in the file. a high level of duplication could indicate a enrichment bias (i.e. PCR amplification). Low level of duplication may indicate a very high level of coverage of the target sequence.</p></li>
<li><p><strong>Overrepresented sequences</strong>: show in a single sequence is very overrepresented in the file. This could indicate a contamination or a problem in the library preparation.</p></li>
<li><p><a href="#id1"><span class="problematic" id="id2">**</span></a>Adapter content <a href="#id3"><span class="problematic" id="id4">**</span></a>: shows the presence of adapter sequences in the reads. If there is presence of adapters, the reads should be trimmed before further analysis.</p></li>
</ol>
</div></blockquote>
<div class="admonition seealso">
<p class="admonition-title">See also</p>
<p>For more information about FASTQC modules interpretation visit the <a class="reference external" href="https://www.bioinformatics.babraham.ac.uk/projects/fastqc/Help/3%20Analysis%20Modules/">FASTQC</a> website.</p>
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</ul>
<p>After preprocessing our reads, its important to check again the Quality. Fastp generates both htm and json report for asses the quality of our reads.
The json reports could be aggregated with MULTIQC.</p>
<p>Example of fastp report.</p>
</section>
</section>
</section>
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