TITLE: Seek & Blastn Standard Operating Procedure
AUTHORS: Jennifer A. Byrne, Yasunori Park, Amanda Capes-Davis, Bertrand Favier, Guillaume Cabanac, Cyril Labbé

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Seek & Blastn is a semi-automated tool that automatically extracts gene identifiers and nucleotide sequences using named entity recognition techniques. The sentence containing each sequence is automatically analyzed to assign a claimed status (targeting or nontargeting) that is compared with the most likely status according to blastn analysis using the human genomic + transcript database. Subsequently, this tool can be used to identify nucleotide sequence reagent errors in the published or forthcoming scientific literature. </div><div class = "text-block">The Seek & Blastn tool was developed by Cyril Labbé from the University of Grenoble Alpes, The National Center for Scientific Research (CNRS), Grenoble, France. </div><div class = "text-block">The authors gratefully acknowledge grant funding from the US Office of Research Integrity, grant ID ORIIR180038-01-00 (awarded to Jennifer Byrne and Cyril Labbé) and the National Health and Medical Research Council of Australia, Ideas grant ID APP1184263 (awarded to Jennifer Byrne, Cyril Labbé and Amanda Capes-Davis).</div><div class = "text-block">To cite Seek & Blastn or to find further information regarding the tool, please refer to:    </div><div class = "text-block"><span>- Labbé C, Grima N, Gautier T, Favier B, Byrne JA. Semi-automated fact-checking of nucleotide sequence reagents in biomedical research publications: The Seek & Blastn tool. PLoS One. 2019 Mar 1;14(3):e0213266. doi: </span><a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213266" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1371/journal.pone.0213266</span></a><span>. PMID: </span><a href="https://pubmed.ncbi.nlm.nih.gov/30822319/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">30822319</span></a><span>; PMCID: </span><a href="https://europepmc.org/article/pmc/pmc6396917" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PMC6396917</span></a><span>.</span></div><div class = "text-block">For further information regarding this tool: </div><div class = "text-block"><span>- Byrne, J.A., Labbé, C. Striking similarities between publications from China describing single gene knockdown experiments in human cancer cell lines.</span><span style = "font-style:italic;">Scientometrics </span><span style = "font-weight:bold;">110,</span><span>1471–1493 (2017). </span><a href="https://link.springer.com/article/10.1007/s11192-016-2209-6#citeas" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s11192-016-2209-6</span></a><span></span></div><div class = "text-block"><span>- Phillips N. Online software spots genetic errors in cancer papers. Nature. 2017 Nov 20;551(7681):422-423. doi: </span><a href="https://www.nature.com/news/online-software-spots-genetic-errors-in-cancer-papers-1.23003" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1038/nature.2017.23003</span></a><span>. PMID: </span><a href="https://pubmed.ncbi.nlm.nih.gov/29168818/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">29168818</span></a><span style = ":;">.</span></div><div class = "text-block"><span>- Byrne JA, Grima N, Capes-Davis A, Labbé C. The Possibility of Systematic Research Fraud Targeting Under-Studied Human Genes: Causes, Consequences, and Potential Solutions. Biomark Insights. 2019 Feb 5;14:1177271919829162. doi: </span><a href="https://journals.sagepub.com/doi/10.1177/1177271919829162" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1177/1177271919829162</span></a><span>. PMID: </span><a href="https://pubmed.ncbi.nlm.nih.gov/30783377/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">30783377</span></a><span>; PMCID: </span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366001/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PMC6366001</span></a><span>.</span></div><div class = "text-block"><span>- Byrne JA, Christopher J. Digital magic, or the dark arts of the 21</span><span style = "vertical-align:super;">st</span><span>century-how can journals and peer reviewers detect manuscripts and publications from paper mills? FEBS Lett. 2020 Feb;594(4):583-589. doi: </span><a href="https://febs.onlinelibrary.wiley.com/doi/full/10.1002/1873-3468.13747" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1002/1873-3468.13747</span></a><span>. Epub 2020 Feb 17. PMID: </span><a href="https://pubmed.ncbi.nlm.nih.gov/32067229/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">32067229</span></a><span style = ":;">.</span></div><div class = "text-block"><span>- Labbé, C., Cabanac, G., West, R.A.</span><span style = "font-style:italic;">et al. </span><span>Flagging incorrect nucleotide sequence reagents in biomedical papers: To what extent does the leading publication format impede automatic error detection?.</span><span style = "font-style:italic;">Scientometrics </span><span style = "font-weight:bold;">124,</span><span>1139–1156 (2020). </span><a href="https://link.springer.com/article/10.1007%2Fs11192-020-03463-z" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s11192-020-03463-z</span></a><span></span></div></div>

[STEPS]
?. [Upload publication to Seek & Blastn]
Upload the publication of interest in pdf format to Seek & Blastn
?. [Upload publication to Seek & Blastn]
For Seek & Blastn, click here.Click on the above link and upload the publication of interest by selecting browse, then submit. In this procedure, the retracted article: PMID 25893892 will be used as an example. Multiple publications may be uploaded at once by compressing the pdf of publications into a zip file and uploading the zip file.
?. [Upload publication to Seek & Blastn]
Wait for Seek & Blastn to finish analyzing the publication, indicated when the window presents the phrase “All done: results_here.” Click on the hyperlink to be taken to the Seek & Blastn output page.
?. [Interpreting Seek & Blastn outputs]
The output of a Seek & Blastn analysis is presented in tabular form divided into six columns, as shown below for the retracted publication PMID 25893892.
?. [Interpreting Seek & Blastn outputs]
The first column (titled Tested file) provides two links as follows: A) a link to the XML file of the uploaded pdfB) a link to the uploaded pdf file analyzed by Seek & Blastn.
?. [Interpreting Seek & Blastn outputs]
The second column (titled Similarity) shows the results of intertextual distance analysis (Labbé & Labbé 2012). The PMID of the most textually similar publication in the reference corpus  (Byrne & Labbé 2017) is provided with the intertextual distance analysis result, divided into the following similarity categories: "OK", "close" and "very close".
Intertextual distance describes the degree of textual similarity between the query and the most similar publication in a reference publication corpus. This distance is provided as a value from 0 to 1, where smaller values indicate greater textual similarity. (Labbé & Labbé 2012)The degree of similarity for: "OK" represents an intertextual distance of > 0.5 to the nearest reference publication. "Close" represents an intertextual distance  0.44 - 0.50 to the nearest reference publication as above. "Very close" represents an intertextual distance of Values were based on the intertextual distance score distribution of 4094 publications from the International Journal of Clinical and Experimental Medicine (Byrne & Labbé 2017)
?. [Interpreting Seek & Blastn outputs]
The third, fourth, and fifth columns (titled Genes, Cont. CL, and Species, respectively) list identifiers detected by Seek & Blastn within the uploaded publication. If no identifiers are found by Seek & Blastn, the relevant column is left blank. "Genes" lists individual gene identifiers (GeneCards) from the query publication, with the number of times that Seek & Blastn recognized the identifier shown in brackets.  "Cont. CL" lists any cell line (CL) identifier that has been associated with contamination or misidentification according to the database of Cross-contaminated or Misidentified Cell Lines version 7.2 established by the International Cell Line Authentication Committee (ICLAC). "Species" lists species identifiers such as "human", "mouse", "rabbit" (Linnaeus) with the number of times each identifier is detected shown in brackets.
It should be noted by users the ICLAC list of cell lines continues to be updated, and the version implemented in Seek & Blastn (version 7.2, 6 October 2014) is not the latest version (see https://iclac.org/databases/cross-contaminations/).
Uploaded publications are considered to describe human research if the frequency of the term "human" is ≥ (the number of times the most frequent species "not human" -1) mentioned in the publication.In publications where this criterion is not met (i.e. "human" In publications that describe the use of human cancer cell lines, other species can be mentioned in association with antibodies. These species may cause Seek & Blastn to not extract sequences and skip the paper. In this case, go to step 4.1.
?. [Interpreting Seek & Blastn outputs (Sequences)]
The sixth column (titled "Sequences.html") lists the Blastn results for all the nucleotide sequences that were extracted by Seek & Blastn. Individual sequences are listed in rows.
?. [Interpreting Seek & Blastn outputs (Sequences)]
The gene symbols and their synonyms are the identifiers associated by Seek & Blastn with the extracted sequence within the uploaded publication. These are from "The approved symbol and synonyms list established by the HUGO Gene Nomenclature Committee (HGNC)". When no gene symbol is extracted by Seek & Blastn, it is indicated by empty brackets (...).
?. [Interpreting Seek & Blastn outputs (Sequences)]
Extracted nucleotide sequences are provided as blue hyperlinks, that when clicked, runs a Google Scholar search using the nucleotide sequence as a query (figure 3.3). Next to each sequence is a "Text" hyperlink that aims to identify the location of the extracted sequence within the text. It searches the first three nucleotides of the sequence in the pdf and thereby may not directly lead to the sequence.
?. [Interpreting Seek & Blastn outputs (Sequences)]
Next to the text claims (step 3.4) is the gene name corresponding to each targeting Blastn hit that is hyperlinked to the associated Blastn results, shown below in figure 3.5. This can support manual validation of results, as per step 5. Where a nucleotide sequence is found to be non-targeting in a Blastn search, this is indicated by "No clear target".
?. [Interpreting Seek & Blastn outputs (Sequences)]
Where the gene symbol of an extracted sequence matches a Blastn search, the integer to the left of the gene symbol remains black. When a gene symbol however does not match any of the significant Blastn hits, the number is highlighted in red, indicating a possible mismatch between the extracted nucleotide sequence and its claimed identity (figure 3.2). It is recommended that results flagged with a red integer be manually verified, as per step 5.
Integers next to gene symbols and their respective definitions: -1: Seek & Blastn was unable to identify whether the sequence is targeting or non-targeting0: No error detected6: "Targeting" sequence is predicted to be non-targeting 7: "Non-targeting" sequence is predicted to be targeting 8: Nucleotide sequence reagent is targeting, but is predicted to target the wrong gene or sequence0: No error detected6: "Targeting" sequence is predicted to be non-targeting 7: "Non-targeting" sequence is predicted to be targeting 8: Nucleotide sequence reagent is targeting, but is predicted to target the wrong gene or sequence6: "Targeting" sequence is predicted to be non-targeting 7: "Non-targeting" sequence is predicted to be targeting 8: Nucleotide sequence reagent is targeting, but is predicted to target the wrong gene or sequence7: "Non-targeting" sequence is predicted to be targeting 8: Nucleotide sequence reagent is targeting, but is predicted to target the wrong gene or sequence
?. [Interpreting Seek & Blastn outputs (Sequences)]
Grey hypertext indicates that Seek & Blastn did not detect the targeting/non-targeting status of the sequence. These sequences can be manually verified as per step 5.  Green hypertext indicates that the claimed status agrees with the verified status , either "Claims targeting" or "Claims non-targeting", shown below in figure 3.7. Orange hypertext indicates Blastn hits of lower significance, explained in figure 3.8. If Blastn analyses return only low significance hits, these should be manually validated as per step 5. Red hypertext indicates Blastn results that conflict with the claimed targeting/non-targeting status of the nucleotide sequence (figure 3.9). Claimed targeting sequences lacking clear targets are indicated by “!! No clear target” or “!! No hits found” in red hypertext. Claimed non-targeting sequences retrieving significant Blastn results are indicated by "!! (Blastn hit)". These results can be manually verified as per step 5.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or RT-PCR/PCR primers, excluding miR reagents)]
Where Seek & Blastn flags a possible incorrect status for any nucleotide sequence reagent, we strongly recommend that such sequences are manually verified prior to taking further action. The recommended steps to manually verify a sequence are summarised in figure 5.0.
?. [Validating Seek & Blastn outputs (recommended)]
Seek & Blastn may not retrieve all the relevant information from uploaded publications (Labbé et al. 2019, Labbé et al. 2020). The following steps are recommended to ensure that all Seek & Blastn outputs are analyzed.
?. [Validating Seek & Blastn outputs (recommended)]
Potentially misidentified cell lines flagged by Seek & Blastn can be found on Cellosaurus. Check the context of any flagged cell lines within the publication to determine if they are being used appropriately. Additionally, Cellosaurus can be used to identify ambiguously named cell lines, to determine whether a misidentified cell line is indeed being used in the publication or if it simply shares the same name as a non-problematic cell line. If there are any problematic cell lines used within the wrong context, go to step 7. To verify any nucleotide sequences, go to step 4.2.
?. [Validating Seek & Blastn outputs (recommended)]
Check that all sequences have been extracted by Seek & Blastn. If the number of sequences in Seek & Blastn outputs matches the number of sequences in the publication, go to step 4.3. If not, any skipped sequences can be manually verified as per step 5.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or PCR primers)]
Blastn can be accessed here. Run a Blastn analysis of flagged nucleotide sequence reagents using the parameters: Human genomic plus transcript (Human G+T) database, optimized for somewhat similar sequences, with the expect threshold set to 1000. If a significant hit is obtained for sequence reagents assigned a grey/orange/red hypertext, check if any of the hits match the claim of the sequence made within the publication or of a misidentified sequence. Any significant hits should be crosschecked against potential synonyms in the Genecards database to avoid incorrectly flagging a valid nucleotide sequence.  If a significant hit is obtained that does not match the claimed status or target of the nucleotide sequence nor any synonyms under the Genecards database, it may be flagged as a misidentified nucleotide sequence. If blastn results do not provide any significant hits, proceed to step 5.3.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or PCR primers)]
Repeat the Blastn analysis against the standard database (nucleotide collection (nr/nt), optimized for somewhat similar sequences, with the expect threshold set to 1000. Unrestricted Blastn searches may identify other hits in humans or other species.  Any significant hits obtained that do not match the claimed status or target of the nucleotide sequence reagent nor any synonyms under the Genecards database, may be flagged as a misidentified nucleotide sequence. If there are no significant hits proceed to step 5.4.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or PCR primers)]
If the query sequence is claimed to target a human gene, the sequence can be used as a query in the UCSC Genome Browser. 1. The genomic location of the claimed target can be found via Genecards for both hg38 and hg19 (i.e. the chromosome and the arm). 2. Open the BLAT search genome available on the UCSC genome browser. Copy the query sequence in the search using the following parameters: Genome: humanAssembly: Grch38/hg38  (If the publication references a different assembly, that should be used here)Query type: BLAT's guess Sort output: Query, scoreOutput type: Hyperlink. 3. If a result is available, the location of the sequence can be viewed in either the "browser" (adjacent to the search bar) or "details" (under "Side by Side Alignment") option. There are several possibilities from a search of the UCSC Genome Browser. If a genomic hit is identified within the range of the claimed target as specified by Genecards, the claimed status may be correct.  If the wrong location appears, the sequence may still be targeting but the wrong gene. If no result appears, proceed to step 5.5
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or PCR primers)]
The query sequence can be searched for in Google Scholar (step 3.3.)Check whether the claimed sequence is used for the same purpose within other publications to determine if the sequence had the correct claim of being a non-targeting or targeting reagent. In the case the sequence is a targeting sequence, it should be checked whether it does target the right sequence. If the sequence either does not return any results as a query on Google Scholar or is used solely as a non-targeting reagent within other publications, it is accepted as a non-targeting sequence.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or PCR primers)]
Thereby, a summary of the workflow for verification of Seek & Blastn results is as follows: A "correct" match can be seen from step 5.2. or 5.3. if the claimed identity of the sequence appears as a significant hit in a Blastn search orIf the location of the sequence in the human genome matches the claim as per step 5.4.A "wrong"/mismatch between the claim and the identity of the sequence is from:step 5.2 or 5.3. if a significant hit contradicting the claim of the publication appears in a Blastn search orIf the location of the sequence in the human genome differs from the claim as per step 5.4. orIf the sequence is revealed to not be used for the same purpose as other publications via a Google Scholar searchWhereas an identification of a non-targeting sequence is from:step 5.2 and 5.3. if there is no significant hit from either Blastn search andIf the sequence does not return any hits for a location in the human genome as per step 5.4. and If the sequence does not provide any hits in a Google Scholar search or is used by other publications as a non-targeting control. A "wrong"/mismatch between the claim and the identity of the sequence is from:step 5.2 or 5.3. if a significant hit contradicting the claim of the publication appears in a Blastn search orIf the location of the sequence in the human genome differs from the claim as per step 5.4. orIf the sequence is revealed to not be used for the same purpose as other publications via a Google Scholar searchWhereas an identification of a non-targeting sequence is from:step 5.2 and 5.3. if there is no significant hit from either Blastn search andIf the sequence does not return any hits for a location in the human genome as per step 5.4. and If the sequence does not provide any hits in a Google Scholar search or is used by other publications as a non-targeting control. Whereas an identification of a non-targeting sequence is from:step 5.2 and 5.3. if there is no significant hit from either Blastn search andIf the sequence does not return any hits for a location in the human genome as per step 5.4. and If the sequence does not provide any hits in a Google Scholar search or is used by other publications as a non-targeting control.
?. [Validating Seek & Blastn outputs (recommended)]
To manually validate a nucleotide sequence reagent for gene knockdown, PCR, or RT-PCR of a gene other than a microRNA (miR), go to step 5.To manually validate a reverse RT-PCR primer or RT reagent designed for analyzing a miR, got to step 6.
?. [Interpreting Seek & Blastn outputs (Sequences)]
Next to each "Text" link are the detected corresponding text claims, shown as either “Claims targeting”, “Claims non-targeting”, or “Undetected”. These claims can be verified as per step 5. If a sequence instead has the message “(Seq. not correctly extracted (Char/long/ short)”, go to step 4.2. If Seek & Blastn is able to identify a sequence as either targeting or non-targeting, outputs will be provided in either green/orange/red hypertext (see step 3.6) If Seek & Blastn is unable to detect a claim, the output hypertext is shown in grey (step 3.6).Outputs shown in grey, orange, or red hypertext can be verified as per step 5.
?. [Manual verification of sequence identity ]
To confirm a Seek & Blastn result, visually inspect the publication to determine whether the sequence's identity is specified to be targeting or non-targeting within the text.
?. [Manual verification of sequence identity ]
Additionally confirm if these reagents were used to analyse human gene or human genomic sequences.
?. [Manual verification of sequence identity ]
?. [Manual verification of sequence identity ]
?. [Manual verification of sequence identity ]
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or RT-PCR/PCR primers, excluding miR reagents)]
Visually inspect the publication to determine that the flagged sequence is claimed to be targeting or non-targeting within the text. 5.1.1. If the text claim for the sequence was incorrectly extracted by Seek & Blastn, check whether the claim matches the Blastn result from Seek & Blastn.           5.1.1.1. If the text claim matches the Seek & Blastn result in full (e.g., targeting the correct genetic target) accept the reagent as correctly identified.           5.1.1.2. If the text claim does not match the Seek & Blastn result in full (wrong targeting status or genetic target), go to step 5.2          5.1.2. If the claim was correctly extracted by Seek & Blastn, the sequence may be wrongly identified within the text. In this case, go to step 5.2.           5.1.1.1. If the text claim matches the Seek & Blastn result in full (e.g., targeting the correct genetic target) accept the reagent as correctly identified.           5.1.1.2. If the text claim does not match the Seek & Blastn result in full (wrong targeting status or genetic target), go to step 5.2If a nucleotide sequence reagent is claimed to target a species other than human, the genetic target and species can be confirmed in step 5.4.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or RT-PCR/PCR primers, excluding miR reagents)]
Blastn can be accessed here. Run a Blastn analysis for each flagged nucleotide sequence reagent using the following parameters for claimed human sequences: Human genomic plus transcript (Human G+T) database, optimized for somewhat similar sequences, with the "expect threshold" set to 1000. 5.2.1. If the Blastn results suggest that the query is a targeting sequence (step 3.4), check whether the Blastn results match the claimed identity within the text.           5.2.1.1. If the Blastn results match the claimed identity, the sequence can be accepted as targeting the claimed target. No further action is needed.           5.2.1.2. If the Blastn results do not match the claimed identity, the query may target another gene or target. In this case, potential recommended courses of action are provided in step 7.Where nucleotide sequence reagents are suggested to target another gene or target, gene identifiers should be crosschecked against potential synonyms in the Genecards (Genecards) database to avoid incorrectly flagging a valid targeting sequence. 5.2.2. If the query does not return any significant results in a Blastn search, proceed to step 5.3.           5.2.1.1. If the Blastn results match the claimed identity, the sequence can be accepted as targeting the claimed target. No further action is needed.           5.2.1.2. If the Blastn results do not match the claimed identity, the query may target another gene or target. In this case, potential recommended courses of action are provided in step 7.Where nucleotide sequence reagents are suggested to target another gene or target, gene identifiers should be crosschecked against potential synonyms in the Genecards (Genecards) database to avoid incorrectly flagging a valid targeting sequence.
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or RT-PCR/PCR primers, excluding miR reagents)]
If the query sequence is claimed to target a human gene or genomic sequence, the sequence can be used to query the UCSC Genome Browser. 5.3.1. The genomic location (i.e. the chromosome and the arm) of the claimed target can be found via both the UCSC Genome Browser or Genecards for the human genome assembly GRCh38/hg38 or for a specified version such as GRCh37/hg19. 5.3.2. Open the BLAT search genome available on the UCSC genome browser and copy in the query sequence. Use the following search parameters:          Genome: human          Assembly: GRCh38/hg38 (or otherwise as stated by the publication)          Query type: BLAT's guess           Sort output: Query, score          Output type: Hyperlink 5.3.3. If the query returns a hit, the location can be viewed in either the "browser" (adjacent to the search bar) or "details" (under the "Side by Side Alignment") option.           5.3.3.1. If a hit is identified within the genomic range of the claimed target, available within the genome browser, the claimed status may be correct. No further action may be needed.           5.3.3.2. If a genomic hit is identified at a location outside the range of the claimed target, the sequence may be targeting a different target. In this case, potential recommended courses of action are provided in step 7.5.3.4. If no result appears, indicated by the BLAT search returning the message "Sorry, no matches found", in either GRCh38 or GRCh37, proceed to step 5.4. 5.3.2. Open the BLAT search genome available on the UCSC genome browser and copy in the query sequence. Use the following search parameters:          Genome: human          Assembly: GRCh38/hg38 (or otherwise as stated by the publication)          Query type: BLAT's guess           Sort output: Query, score          Output type: Hyperlink 5.3.3. If the query returns a hit, the location can be viewed in either the "browser" (adjacent to the search bar) or "details" (under the "Side by Side Alignment") option.           5.3.3.1. If a hit is identified within the genomic range of the claimed target, available within the genome browser, the claimed status may be correct. No further action may be needed.           5.3.3.2. If a genomic hit is identified at a location outside the range of the claimed target, the sequence may be targeting a different target. In this case, potential recommended courses of action are provided in step 7.5.3.4. If no result appears, indicated by the BLAT search returning the message "Sorry, no matches found", in either GRCh38 or GRCh37, proceed to step 5.4.           5.3.3.1. If a hit is identified within the genomic range of the claimed target, available within the genome browser, the claimed status may be correct. No further action may be needed.           5.3.3.2. If a genomic hit is identified at a location outside the range of the claimed target, the sequence may be targeting a different target. In this case, potential recommended courses of action are provided in step 7.          5.3.4.1. This may result from the query being too short ("Warning: Sequence YourSeq is only [a value ] letters long (20 is the recommended minimum)")
?. [Manual verification of Seek & Blastn results (Gene knockdown and/or RT-PCR/PCR primers, excluding miR reagents)]
Sequences found to be non-targeting in both the Blastn G+T database (step 5.2) and the UCSC BLAT search genome (step 5.3.) can be used as queries in a Blastn analysis, against the standard database (nucleotide collection (nr/nt)), optimized for somewhat similar sequences, with the "expect threshold" set to 1000. Unrestricted Blastn searches may identify hits in other species. 5.4.1. If Blastn suggests the query sequence is targeting, check whether the species and genetic target match the text claim.            5.4.1.1. If both the species and genetic identity match the text claim, the query can be accepted as a correct targeting reagent.           5.4.1.2. As described in step 5.2.1, if Blastn results do not match the claimed status or target, nor any synonyms under the Genecards database, the query may be non-targeting in the claimed species. Potential recommended courses of action are provided in step 7.5.4.2. If there are no significant Blastn hits, the query can be accepted as a non-targeting sequence. If the sequence was claimed to be non-targeting, no further action is needed.            5.4.1.1. If both the species and genetic identity match the text claim, the query can be accepted as a correct targeting reagent.           5.4.1.2. As described in step 5.2.1, if Blastn results do not match the claimed status or target, nor any synonyms under the Genecards database, the query may be non-targeting in the claimed species. Potential recommended courses of action are provided in step 7.          5.4.2.1. If the sequence was claimed to be targeting but is found to be non-targeting, recommended courses of action are provided in step 7.
?. [Manual verification of Seek & Blastn results (analysing reverse transcriptase (RT) primers and RT-PCR primers for miR analysis)]
Amplification of miRs via RT-PCR typically involves the addition of a 3' sequence using an RT primer and the use of a reverse RT-PCR primer that binds only the 3' 4-8 nucleotides of the target miR (Dellett and Simpson, 2016). Consequently, RT-PCR miR reagents are more likely to be flagged by Seek & Blastn as non-targeting.
?. [Recommended steps in the event of incorrect nucleotide sequence reagents]
After the identification of an incorrect nucleotide sequence reagent or problematic cell line, possible further actions include:Publications can be commented on in PubPeer (https://pubpeer.com/).
?. [Manual verification of Seek & Blastn results (analysing reverse transcriptase (RT) primers and RT-PCR primers for miR analysis)]
miR RT-PCR forward primers can be verified as per step 5.2, and/or as a query in miRBase. 6.1.1. If the 3' end of a miR forward RT-PCR primer shows 100% sequence identity to the claimed miR, over at least 12 consecutive ribonucleotides (Busk 2014), it can be accepted as targeting.
?. [Manual verification of Seek & Blastn results (analysing reverse transcriptase (RT) primers and RT-PCR primers for miR analysis)]
To analyze miR RT primers, retrieve the relevant miR sequence from miRBase. If the miR RT primer is not provided, go to step 6.3. 6.2.1. If the miR strand is specified (e.g., "hsa-miR-145-5p"), only the relevant strand needs to be analyzed.           6.2.1.1. If the specific miR strand is not specified (e.g., "hsa-miR-145"), analyze both the 5p and 3p strands of the miR. 6.2.2. Manually align the nucleotides on the 3' end of the RT primer to the 3' end of the claimed miR target.           6.2.1.1. If the specific miR strand is not specified (e.g., "hsa-miR-145"), analyze both the 5p and 3p strands of the miR.           6.2.2.1. If the  RT primer shows at least 4 consecutive nucleotides of complementarity, including the 3' end of the miR target (Balcells et al., 2011), the RT primer is accepted as a targeting reagent.           6.2.2.2. If the RT primer does not show at least 4 consecutive nucleotides of complementarity, including the 3' end of the miR target, the RT primer is unlikely to bind the miR target and may be non-targeting. In this case, potential courses of action are provided in step 7.
?. [Manual verification of Seek & Blastn results (analysing reverse transcriptase (RT) primers and RT-PCR primers for miR analysis)]
miR reverse RT-PCR primers are analyzed as in step 6.2. Where an RT primer is provided, the reverse RT-PCR primer can also be compared to the RT primer as well. 6.3.1. If the reverse RT-PCR primer has 100% sequence identity to the RT primer over at least 17 consecutive nucleotides, it is likely to represent a targeting reagent, assuming the RT primer binds the miR as per step 6.2.2.1.6.3.2. If the miR RT-PCR reverse primer does not share 100% sequence identity with the RT primer over at least 17 consecutive nucleotides, it may represent a non-targeting reagent. Potential courses of action are provided in step 7.6.3.3. If the RT primer sequence is not provided, manually align the 3' end of the reverse RT-PCR primer to the 3' end of the claimed miR target.           6.3.3.1. If the reverse RT-PCR primer shows, as per step 6.2.2.1 at least 4 consecutive nucleotides that are complementary to the 3' end of the miR target (Balcells et al., 2011), it is likely to represent a targeting reagent.           6.3.3.2. If the reverse RT-PCR primer shows step 6.3.4.  6.3.4. If the RT primer is not provided, and the reverse RT-PCR primer 3' end is not indicated to bind to the claimed miR target, use the reverse RT-PCR primer as a query in Google Scholar (figure 3.3 B)6.3.2. If the miR RT-PCR reverse primer does not share 100% sequence identity with the RT primer over at least 17 consecutive nucleotides, it may represent a non-targeting reagent. Potential courses of action are provided in step 7.6.3.3. If the RT primer sequence is not provided, manually align the 3' end of the reverse RT-PCR primer to the 3' end of the claimed miR target.           6.3.3.1. If the reverse RT-PCR primer shows, as per step 6.2.2.1 at least 4 consecutive nucleotides that are complementary to the 3' end of the miR target (Balcells et al., 2011), it is likely to represent a targeting reagent.           6.3.3.2. If the reverse RT-PCR primer shows step 6.3.4.  6.3.4. If the RT primer is not provided, and the reverse RT-PCR primer 3' end is not indicated to bind to the claimed miR target, use the reverse RT-PCR primer as a query in Google Scholar (figure 3.3 B)6.3.3. If the RT primer sequence is not provided, manually align the 3' end of the reverse RT-PCR primer to the 3' end of the claimed miR target.           6.3.3.1. If the reverse RT-PCR primer shows, as per step 6.2.2.1 at least 4 consecutive nucleotides that are complementary to the 3' end of the miR target (Balcells et al., 2011), it is likely to represent a targeting reagent.           6.3.3.2. If the reverse RT-PCR primer shows step 6.3.4.  6.3.4. If the RT primer is not provided, and the reverse RT-PCR primer 3' end is not indicated to bind to the claimed miR target, use the reverse RT-PCR primer as a query in Google Scholar (figure 3.3 B)          6.3.3.1. If the reverse RT-PCR primer shows, as per step 6.2.2.1 at least 4 consecutive nucleotides that are complementary to the 3' end of the miR target (Balcells et al., 2011), it is likely to represent a targeting reagent.           6.3.3.2. If the reverse RT-PCR primer shows step 6.3.4.           6.3.3.1. If the reverse RT-PCR primer has been used as a reverse RT-PCR primer for other miRs, or has been described as a universal primer, it may represent an appropriate reverse RT-PCR primer. However, it's possible that it's stated genetic identity (as a reverse RT-PCR primer for a specific miR) is incorrect.            6.3.3.2. If the reverse RT-PCR primer has not been used as a reverse RT-PCR primer by other publications, or listed as a universal primer, it may represent a non-targeting reagent. Potential courses of action are provided in step 7.
?. [Manual verification of Seek & Blastn results (analysing reverse transcriptase (RT) primers and RT-PCR primers for miR analysis)]
An example of an analysis of miR RT-PCR reagents is shown in figure 6.0. An example of an analysis of miR RT-PCR reagents shown to be incorrect is shown in figure 6.1.
?. [Validating Seek & Blastn outputs (recommended)]
Check that all sequences have been extracted correctly. If a nucleotide sequence is either  91 nucleotides, Seek & Blastn will recognize this sequence as being incompletely or incorrectly extracted and will flag the reagent with “(Seq. not correctly extracted (Char/long/ short)”. If any sequences have been flagged by Seek & Blastn or incorrectly extracted, manually copy the sequence from the publication and verify, as per step 5.